TWI853843B - Bioreactor and method for scaling production of cells - Google Patents

Abstract

The present disclosure contemplates components, systems and methods for bioreactors that may be employed for producing and maintaining cells, optimizing cell growth and production of products from such cells, and for producing and isolating cells and products made by such cells. The systems, components and methods herein address the scale, cost, efficiency and consistency and are suitable for bespoke cell and bioproduct production.

Description

Bioreactor and method for large-scale production of cells

The present invention relates to systems, components and methods for producing and maintaining cells and for producing and isolating cells and products made from such cells.

The production of bioproducts including cells, proteins and small and large chemical molecules has become a growing focus in the provision of medical, food, industrial and other types of products. Product consistency and the ability to scale production as well as the flexibility to customize manufacturing for different locations and environmental conditions are important factors in production.

Bioreactors provide an environment for the large-scale production of cells and the production of proteins and other molecules from these cells. Many bioreactors require large capital investments and require large physical spaces. In addition, the environment of large bioreactors can differ from that of smaller-scale growth chambers and thereby result in suboptimal growth and production conditions. The large scale of bioreactors can also make it difficult to study growth conditions at the individual cell level. This can result in population heterogeneity of cells and affect the quality, purity, and yield of the bioproducts produced by the cells.

Provided herein are systems, components and methods for producing and maintaining cells and for producing and isolating cells and products made from these cells. The systems, components and methods herein address scale, cost, efficiency and consistency.

In one aspect, the present invention provides a bioreactor comprising: an inlet configured to receive a plurality of cells; a plurality of mini-modules fluidically connected to the inlet, wherein the mini-modules of the plurality of mini-modules comprise a double helical tetracosahedron structure or a modified double helical tetracosahedron structure, wherein the plurality of mini-modules are fluidically interconnected to provide at least one microchannel configured to allow the plurality of cells to flow; and an outlet fluidically connected to the plurality of mini-modules, the outlet being configured to guide the plurality of cells or their derivatives out of the at least one microchannel.

In some embodiments, the mini-modules are interconnected in a manner that provides at least two non-overlapping microchannels each having a constant mean curvature. In some embodiments, a first microchannel of the at least two non-overlapping microchannels is configured to flow a liquid medium, and wherein a second microchannel of the at least two non-overlapping microchannels is configured to flow a gas composition. In some embodiments, the at least two non-overlapping microchannels provide a liquid. In some embodiments, the at least two non-overlapping microchannels are separated by a porous membrane. In some embodiments, the area of the first microchannel is equivalent to the area of the second microchannel, and wherein the area of the porous membrane is the sum of the areas of the first microchannel and the second microchannel. In some embodiments, a plurality of mini-modules are assembled into a macrostructure. In some embodiments, the macrostructure is selected from the group consisting of a pyramid, a hollow pyramid, a thin pyramid, a thin sheet, a checkerboard arrangement, and a log. In some embodiments, a plurality of mini-modules are arranged in a layer within the macrostructure, and wherein the layers are configured such that the velocity of the liquid medium in each layer is substantially the same. In some embodiments, a plurality of mini-modules are arranged in a layer within the macrostructure, and wherein the layers are configured such that the velocity of the liquid medium varies within the layer. In some embodiments, the liquid medium flowing through the at least one microchannel has a velocity greater than the free fall velocity of the cells flowing through the at least one microchannel. In some embodiments, the bioreactor further comprises a gas input port at the base of the macrostructure and a gas output port at the top of the macrostructure. In some embodiments, the bioreactor further comprises a cell input port at the top of the macrostructure configured to provide a plurality of cells, and a cell collection device at the base of the macrostructure configured to collect a plurality of cells. In some embodiments, the bioreactor further comprises a liquid medium input device configured to flow liquid medium to each layer of the plurality of mini-modules. In some embodiments, the volume of liquid medium provided to each layer by the liquid medium device maintains a substantially constant cell density in each layer. In some embodiments, the rate of liquid medium passing through each mini-module is determined by the cell division rate, so that the time for a cell to pass through a single mini-module or a layer of mini-modules is substantially the same as or proportional to the cell division rate. In some embodiments, the bioreactor is interconnected with the sandbox module. In some embodiments, the bioreactor is interconnected with the cell chip module.

In another aspect, the present invention provides a system for cell production, comprising: a first module, comprising a cell chip configured to accommodate a plurality of cells; a second module, which is fluidically connected to the first module, wherein the second module comprises a sandbox bioreactor, the sandbox bioreactor being configured to (i) interface with the cell chip, (ii) direct cell subsets from the plurality of cells to different segments, wherein the different The cell growth conditions in the same segment can be individually configured, and (iii) a set of growth conditions for the plurality of cells is repeatedly generated; and a third module is fluidly connected to the first module and the second module, wherein the third module includes a bioreactor, which is configured to (i) interface with the second module, (ii) receive the cell subset, and (iii) generate a copy of the cell subset under the set of growth conditions.

In some embodiments, the first module, the second module, and the third module are interconnected via fluid. In some embodiments, the system further includes a pump corresponding to each module, wherein the pump is configured to provide liquid culture medium according to the flow rate or pressure used for the corresponding module. In some embodiments, the pump is a syringe pump, a peristaltic pump, or a pressure pump. In some embodiments, the system further includes a component selected from a group consisting of a culture medium preparer, an electroporator, a storage device, a pump, a bubble sensor, a bubble trap, and a combination thereof. In some embodiments, the system further includes at least one sensor. In some embodiments, the at least one sensor is an online sensor. In some embodiments, the at least one sensor measures a biological parameter, a physical parameter, or a chemical parameter. In some embodiments, the biological parameter is selected from the group consisting of cell division rate, cell growth rate, cell stress response, cell protein content, cell carbohydrate content, cell lipid content, and cell nucleic acid content. In some embodiments, the physical parameter is selected from the group consisting of cell size, cell density, cell flow rate, liquid medium flow rate, mixing rate, turbidity, temperature, and pressure. In some embodiments, the chemical parameter is selected from the group consisting of pH, liquid medium composition, concentration of individual liquid medium components, gas composition and gas concentration, and dissolved gas concentration. In some embodiments, the system further includes a camera device. In some embodiments, the camera device is configured to count cells from a sandbox bioreactor or an output of a bioreactor. In some embodiments, the camera device is configured to capture at least one additional parameter associated with individual cells from the output of at least one bioreactor module, and wherein the additional parameter is a biological, chemical or physical characteristic of the cell.

In another aspect, the present invention provides a cell chip module, comprising: a layered structure including at least one fluid loop; a cell holding region in fluid communication with the at least one fluid loop, wherein the cell holding region includes at least one first capture device configured to hold a plurality of cells; an inlet port in fluid communication with the layered structure and configured to input liquid culture medium into the cell holding region; and an outlet port in fluid communication with the layered structure and configured to collect used or excess culture medium and cells.

In some embodiments, at least one fluid loop is configured to flow gas to the cell holding area. In some embodiments, at least one fluid loop is configured to flow liquid medium to the cell holding area. In some embodiments, at least one trap includes a suction trap. In some embodiments, at least one trap includes a gate trap. In some embodiments, the cell holding area further includes a second trap. In some embodiments, the second trap is an overflow trap. In some embodiments, the at least one trap and the second trap are connected in series with each other. In some embodiments, the cell holding area includes at least one gate trap and two or more overflow traps. In some embodiments, the cell chip module further comprises one or more cells in a storage mode. In some embodiments, the storage mode is selected from cells that are dried, lyophilized, frozen, or suspended in a liquid. In some embodiments, the cell chip module further comprises one or more physical barriers for distributing cells flowing through the cell chip module.

In another aspect, the present invention provides a sandbox bioreactor module comprising a series of segments, wherein one segment of the series of segments comprises at least two microchannels, the at least two microchannels are configured to transport at least one cell from one end of one of the at least two microchannels to the other end of the microchannel of the at least two microchannels, wherein one end of the microchannel is configured to input liquid culture medium and the at least one cell, wherein the other end of the microchannel is configured to output the liquid culture medium and the at least one cell, and wherein the growth conditions in the series of segments can be individually configured.

In some embodiments, the first segment and the second segment of the series of segments are configured in series so that cells are transported from the microchannel of the first segment to the microchannel of the second segment. In some embodiments, the microchannel of the first segment bifurcates at the output end into at least two microchannels from the second segment, wherein the at least two microchannels are configured in a parallel configuration so that cells from the first segment are output to one of the at least two microchannels and another cell from the first segment is output to another of the at least two microchannels. In some embodiments, the sandbox bioreactor further includes a first inlet configured to provide liquid culture medium to the series of segments. In some embodiments, the length of the microchannel is determined by the cell division rate so that the cell divides zero, one, two, three, four, five, or more than five times during the period of transport from one end of the microchannel to the other end of the microchannel. In some embodiments, the diameter of the microchannel is determined by the cell size, the mixing rate of the transported liquid, or a combination thereof. In some embodiments, the sandbox bioreactor further includes at least one sensor for measuring a parameter of the cell environment of the sandbox bioreactor. In some embodiments, the sandbox bioreactor further includes a controller configured to respond to the measurement changes from the at least one sensor to the input of the sandbox bioreactor. In some embodiments, the parameter is selected from the group consisting of biological parameters, physical parameters, and chemical parameters. In some embodiments, the parameter is selected from the group consisting of gas content, gas concentration, pH, optical density, and temperature. In some embodiments, the parameter is selected from the group consisting of cell division rate, cell density, cell stress response, or cell metabolites. In some embodiments, the sandbox bioreactor further comprises a sample collection chamber at the outlet of the microchannel of the last segment in the series of segments. In some embodiments, the sandbox bioreactor is interconnected with the cell chip module.

In another aspect, the present invention provides a method for growing and storing cells, comprising: inoculating a cell chip module with at least one cell, wherein the cell chip module comprises a layered structure having at least one fluid circuit, a cell holding region in fluid communication with the at least one fluid circuit, an inlet port in fluid communication with the layered structure, and a cell holding region in fluid communication with the layered structure. The invention relates to a cell chip and a storage mode for storing the at least one cell in a first capture vessel in the cell holding area; providing a liquid culture medium to the inlet so that the at least one cell is retained in a first capture vessel in the cell holding area; culturing the cell chip for a period of time under conditions sufficient to allow cell division so that the divided cells are retained in the first capture vessel; and placing the cells in a storage mode after the period of cell division.

In some embodiments, the storage mode is selected from the group consisting of drying, lyophilizing, freezing, or suspending in liquid. In some embodiments, the method further comprises providing new liquid medium to the inlet and incubating the cell chip for a period of time under conditions that allow cell division to reactivate cell division. In some embodiments, the cell division period produces enough cells so that a large number of cells leave the first trap and enter the second trap in the cell chip module. In some embodiments, the cells are further incubated for a second period of cell division, and wherein the second period of time produces a sufficient number of cells so that a large number of cells leave the second trap and flow from the cell chip module to the outlet for collection. In some embodiments, cells from the outlet of the cell chip module are provided to an interconnected sandbox or bioreactor module. In some embodiments, the second trap is a suction trap or an overflow trap. In some embodiments, the first trap is a gate trap or a suction trap.

In another aspect, the present invention provides a method for selecting cell growth conditions, comprising: introducing a first group of tissue cells into a sandbox bioreactor comprising a series of segments, wherein one segment of the series of segments can be individually configured; culturing the first group of tissue cells under a first set of growth conditions in a first segment of the series of segments; monitoring a first parameter of the first group of tissue cells in the first segment; and changing the set of growth conditions in response to the monitoring of the first parameter in the first segment to produce a second set of growth conditions in a second segment of the series of segments.

In some embodiments, the first group of cells is moved to the second segment, and the second group of cells is introduced into the first segment. In some embodiments, the first group of cells is introduced into the sandbox bioreactor from the cell chip module. In some embodiments, the sandbox reactor is interconnected with the bioreactor module. In some embodiments, a second set of growth conditions is applied to the bioreactor. In some embodiments, the flow rate of cells from one end of each segment to the other end is determined by the cell division rate. In some embodiments, the cell divides once in the time period in which it is transported from one end of the segment to the other end of the segment. In some embodiments, the liquid medium flow through each segment of the segment series is laminar flow.

In another aspect, the present invention provides a method for large-scale production of cells, comprising: introducing a plurality of cells into an inlet of a bioreactor, wherein the bioreactor comprises a collection of mini-modules of a double helical icosahedron structure or a modified double helical icosahedron structure, wherein the mini-modules are arranged in a layer within a macrostructure comprising an inlet and an outlet; flowing a liquid culture medium into the bioreactor; supplying a gas composition into the bioreactor; and collecting the plurality of cells from the outlet; wherein the plurality of cells are transferred between the mini-modules, and wherein the plurality of cells are transferred from the inlet end of the macrostructure to the outlet end of the macrostructure.

In some embodiments, the plurality of cells divide once on average during transfer from one layer of a mini-module to the next layer of a mini-module. In some embodiments, the amount of liquid medium flowing to each layer of a mini-module maintains substantially the same cell density in each layer. In some embodiments, the velocity of the liquid medium flowing through the bioreactor exceeds the free fall velocity of the cells. In some embodiments, the plurality of cells are introduced into the bioreactor from a cell chip or a sandbox module. In some embodiments, portions of the plurality of cells are collected from an outlet end of a macrostructure. In some embodiments, the plurality of cells produce at least one bioproduct, and wherein the bioproduct is collected from an outlet end of a macrostructure. In some embodiments, the bioproduct is selected from the group consisting of small molecules, proteins, antibodies, large macromolecules, and metabolites.

In another aspect, the present invention provides a method for customized cell production, comprising: introducing cells of a selected type into a cell chip module; growing the cells in the cell chip module; transferring the cells from the cell chip module to a sandbox bioreactor; and selecting at least one growth condition from a first set of growth conditions in the sandbox bioreactor to produce a second set of growth conditions.

In another aspect, the present invention provides a method for culturing cells, comprising: providing a plurality of cells to an adhesion bioreactor comprising at least one channel and a microporous membrane; allowing at least a portion of the plurality of cells to adhere to a surface of the at least one channel so that the at least a portion of the plurality of cells replicates on the surface of the at least one channel to produce attached cells; flowing a liquid culture medium from the at least one channel through the microporous membrane to (i) wash the attached cells, (ii) detach the attached cells to produce suspended cells, (iii) wash the suspended cells; and collect the suspended cells as needed.

In some embodiments, at least one channel includes a material suitable for at least partial adhesion of the plurality of cells. In some embodiments, the method further includes flowing additional liquid medium through the at least one channel to (i) provide medium to allow at least partial growth and/or replication of the plurality of cells, (ii) dissociate attached cells from the at least one channel, or (iii) flow suspended cells from the at least one channel to a collection area. In some embodiments, the adhesion bioreactor is in fluid communication with a cell chip module, and wherein the cell chip module provides the plurality of cells to the adhesion bioreactor. In some embodiments, the adhesion bioreactor is in fluid communication with a bioreactor, and wherein the adhesion bioreactor provides suspended cells to the bioreactor. In some embodiments, the plurality of cells are selected from the group consisting of bacterial cells, fungal cells, yeast cells, eukaryotic cells, plant cells, and algae cells. In some embodiments, the plurality of cells are recombinant cells.

In some embodiments, the method further comprises growing a sample of the selected type of cells in a bioreactor having a second set of growth conditions. In some embodiments, the selected type of cells or a portion thereof is collected from the bioreactor. In some embodiments, the selected type of cells is a chimeric antigen receptor T (CAR-T) cell, a stem cell, or a differentiated cell. In some embodiments, the selected type of cells produces at least one biological product, and wherein the biological product is collected from the bioreactor.

In another aspect, the present invention provides a system comprising a plurality of fluid flow paths having a substantially constant cross-section, wherein a first fluid flow path of the plurality of fluid flow paths is fluidly connected to a second fluid flow path of the plurality of fluid flow paths to allow gas to flow from the first fluid flow path to the second fluid flow path along the first fluid flow path at a substantially constant rate, and wherein the first fluid flow path is configured to allow cell culture.

In some embodiments, the plurality of fluid flow paths include a helical icosahedron structure, a double helical icosahedron structure, a modified double helical icosahedron structure, a triple periodic minimal surface, or a combination thereof.

In another aspect, the present invention provides a method for treating a plurality of cells, comprising: (a) providing a bioreactor, the bioreactor comprising: (i) an inlet; (ii) a plurality of mini-modules fluidically connected to the inlet, wherein the mini-modules of the plurality of mini-modules comprise a double helical icosahedron structure or a modified double helical icosahedron structure, wherein the plurality of mini-modules are fluidically interconnected to provide at least one microchannel; and (iii) an outlet fluidically connected to the plurality of mini-modules; and (b) guiding a plurality of cells to the inlet, the plurality of cells or derivatives thereof being guided from the inlet to the outlet through the at least one microchannel.

In some embodiments, the bioreactor comprises at least two microchannels. In some embodiments, a first microchannel of the at least two microchannels allows a liquid medium to flow. In some embodiments, a second microchannel of the at least two microchannels allows a gas composition to flow. In some embodiments, the at least two microchannels are separated by a porous membrane. In some embodiments, a plurality of mini-modules are assembled into a macrostructure.

In another aspect, the present invention provides a method for producing a bioreactor, the bioreactor comprising: an inlet configured to receive a plurality of cells; a plurality of mini-modules fluidically connected to the inlet, wherein the mini-modules of the plurality of mini-modules comprise a double helical icosahedron structure or a modified double helical icosahedron structure, wherein the plurality of mini-modules are fluidically interconnected to provide at least one microchannel configured to allow the plurality of cells to flow; and an outlet fluidically connected to the plurality of mini-modules, the outlet being configured to guide the plurality of cells or derivatives thereof out of the at least one microchannel.

In some embodiments, the bioreactor is produced using three-dimensional (3-D) printing of a plurality of mini-modules.

In some embodiments, a system for cell production is provided, comprising: a first module comprising a cell chip configured to accommodate a plurality of cells; a second module comprising a sandbox bioreactor configured to (i) interface with the cell chip, (ii) direct cells from the plurality of cells to different segments, wherein cell growth conditions in the different segments can be individually configured, and (iii) repeatedly generate a set of growth conditions for the plurality of cells; and a third module comprising a production bioreactor configured to (i) interface with the second module, (ii) accommodate the cells, and (iii) generate replicates of the cells under the set of growth conditions.

In some embodiments, the first module, the second module, and the third module of the system are functionally interconnected. In some embodiments, the system further includes a pump corresponding to each module, wherein the pump provides liquid culture medium at a flow rate or pressure for the corresponding module. In some embodiments, the pump is a syringe pump, a peristaltic pump, or a pressure pump.

Also provided herein are such systems further comprising a component selected from the group consisting of a medium preparer, an electroporator, a container, a pump, a bubble sensor, a bubble trap, and a combination thereof. In some embodiments, the system also includes at least one sensor, such as an online sensor. In some embodiments, at least one sensor measures a biological parameter, a physical parameter, or a chemical parameter. For example, the biological parameter, the physical parameter, or the chemical parameter may include: a biological parameter selected from the group consisting of cell division rate, cell growth rate, cell stress response, cell protein content, cell carbohydrate content, cell lipid content, and cell nucleic acid content; a physical parameter selected from the group consisting of cell size, cell density, cell flow rate, liquid medium flow rate, mixing rate, turbidity, temperature, and pressure; a chemical parameter selected from the group consisting of pH, liquid medium composition, concentration of individual liquid medium components, gas composition and gas concentration, and dissolved gas concentration; and combinations thereof.

Provided herein are such systems further comprising a camera device. In some embodiments, the camera device counts cells from an output of a sandbox bioreactor or a production bioreactor. In some embodiments, the camera device captures at least one additional parameter associated with individual cells from the output of at least one bioreactor module, wherein the additional parameter is a biological, chemical, or physical characteristic of the cell.

In some embodiments, a cell chip module is provided, which includes: a cell holding area, which includes at least one first trap for holding cells and at least one second trap for holding cells as needed; an inlet port, which is configured to input liquid culture medium into the cell holding area; and an outlet port, which is configured to collect used or excess culture medium and cells. In some embodiments, the first trap can be a suction trap, a gate trap, an overflow trap, or a combination thereof. In some embodiments, the first trap and the second trap are connected in series with each other. In some embodiments, the cell chip module includes at least one gate trap and two or more overflow traps. In some embodiments, a cell chip module may include one or more physical barriers for distributing cells flowing through the module.

In some embodiments, the cell chip module further includes one or more cells in a storage mode, for example, the one or more cells may be dried, cryo-dried, frozen, or suspended in a liquid.

In some embodiments, the present invention provides a sandbox bioreactor module comprising a series of segments, wherein one segment of the series of segments comprises at least one microchannel, the at least one microchannel is configured to transport cells from one end of the microchannel to the other end of the microchannel, and wherein one end of the microchannel is configured to input liquid culture medium and at least one cell, and wherein the other end of the microchannel is configured to output the liquid culture medium and at least one cell.

In some embodiments of the sandbox bioreactor, the first segment and the second segment are configured in series so that cells are transferred from one of the microchannels of the first segment to one of the microchannels of the second segment. In some embodiments, the microchannel of the first segment bifurcates at the output end into at least two microchannels from the second segment, wherein the two microchannels are configured in a parallel configuration so that a cell from the first segment is output into one of the at least two microchannels and another cell from the first segment is output into another of the at least two microchannels.

In some embodiments, the sandbox bioreactor further comprises a first inlet adapted to provide a liquid culture medium to the segment. In some embodiments, the length of each microchannel of the sandbox bioreactor is determined by the cell division rate, such that the cell divides zero, one, two, three, four, five, or more than five times during its transport from one end of the microchannel to the other end of the microchannel. In some embodiments, the diameter of each microchannel is determined by the cell size, the mixing rate of the transported liquid, or a combination thereof.

In some embodiments, the sandbox bioreactor comprises at least one sensor for measuring a parameter of the bioreactor cell environment, and optionally a controller for changing an input to the bioreactor in response to the measurement from the at least one sensor. In some embodiments, the sensor measures a parameter such as a biological parameter, a physical parameter, and/or a chemical parameter such as gas content, gas concentration, pH, optical density, temperature, cell division rate, cell density, cell stress response, cell metabolites, or a combination thereof.

In some embodiments, the sandbox bioreactor comprises a sample collection chamber at the outlet of the microchannel of the last segment in the series of segments. In some embodiments, the sandbox bioreactor is interconnected with a cell chip module (such as any of the cell chip modules described herein).

Provided herein in some embodiments is a production bioreactor comprising a plurality of mini-modules, wherein each of the plurality of mini-modules comprises a double helical tetrahedron shape or structure, and wherein the plurality of mini-modules are interconnected to provide a microchannel. In some embodiments, the interconnection of the mini-modules produces two non-overlapping channels each having a constant mean curvature. In some embodiments, one microchannel of the production bioreactor provides a liquid culture medium and wherein the second microchannel provides a gas composition. In some embodiments, both microchannels provide liquid. In some embodiments, the microchannels are separated by a porous diaphragm. In some embodiments, the area of the first microchannel is equivalent to the area of the second microchannel, and wherein the area of the diaphragm is the sum of the areas of the first microchannel and the second microchannel.

In some embodiments, the production bioreactor may include mini-modules assembled into a macrostructure, for example, the macrostructure may be a pyramid, a hollow pyramid, a thin pyramid, a thin sheet, a checkerboard arrangement, or a log. In some embodiments, the mini-modules are arranged in layers within the macrostructure, and wherein the velocity of the liquid medium in each layer is substantially the same. In some embodiments, the velocity of the liquid medium varies within a layer. In some embodiments, the liquid medium flowing through the microchannel has a velocity greater than the free fall velocity of the cells flowing through the microchannel.

In some embodiments, the production bioreactor includes a gas input port at the base of the macrostructure and a gas output port at the top of the macrostructure. In some embodiments, there is a cell input port at the top of the macrostructure and a cell collection device at the base of the macrostructure.

In some embodiments, the production bioreactor comprises a liquid medium input device, wherein the liquid medium flows into each layer of the mini-module. In some embodiments, the volume of liquid medium provided to each layer by the liquid medium device maintains a substantially constant cell density in each layer. In some embodiments, the speed of liquid medium passing through each mini-module is determined by the cell division rate, so that the time for cells to pass through a single mini-module or mini-module layer is substantially the same as or proportional to the cell division rate.

In some embodiments, the production bioreactor is interconnected with a sandbox module, a cell chip module, or a system comprising both a cell chip module and a sandbox module. In some embodiments, a system is provided herein comprising a plurality of fluid flow paths having a substantially constant cross-section, wherein a first fluid flow path of the plurality of fluid flow paths is fluidly connected to a second fluid flow path of the plurality of fluid flow paths to allow gas to flow from the first fluid flow path to the second fluid flow path along the first fluid flow path at a substantially constant rate. In some embodiments, the plurality of fluid flow paths include a helical icosahedral shape or structure, a modified helical icosahedral shape or structure, a triple periodic minimum surface, or a combination thereof.

Also provided herein are methods for growing and storing cells, comprising: inoculating a cell chip module with at least one cell; providing a liquid culture medium to a liquid inlet of the cell chip such that the at least one cell is retained in a first capture vessel; culturing the cell chip for a period of time under conditions that allow cell division such that the divided cells are retained in the first capture vessel; and after the period of cell division, placing the cells in a storage mode. In some embodiments, the storage mode is selected from the group consisting of drying, lyophilizing, freezing, or suspending in a liquid. In some embodiments, the method further comprises providing new liquid medium to the liquid inlet of the cell chip and incubating the cell chip for a period of time under conditions that allow cell division to reactivate cell division. In some embodiments, the cell division period produces enough cells so that a large number of cells leave the first trap and enter the second trap in the cell chip module. In some embodiments, the cells are further incubated for a second period of time for cell division, and wherein the second period of time for cell division produces enough cells so that a large number of cells leave the second trap and flow from the cell chip to the outlet for collection. In some embodiments, cells from the outlet of the cell chip are provided to an interconnected sandbox or production bioreactor module. In some embodiments of the method, the second trap is a suction trap or an overflow trap. In some embodiments, the first trap is a gate trap or a suction trap.

Provided herein is a method for optimizing a cell environment, comprising: introducing a first group of cells into a sandbox bioreactor comprising a series of segments; cultivating the first group under a first environment in a first segment; monitoring a first parameter of the cells in the first segment; and changing the first environment in response to the monitoring of the first parameter to produce a second environment. In some embodiments of the method, the first group of cells moves to a second segment and wherein the second group of cells is introduced into the first segment. In some embodiments, cells are introduced into a sandbox bioreactor from a cell chip module. In some embodiments, a sandbox reactor is interconnected with a production bioreactor module. In some embodiments of the method, a second environment is applied to the production bioreactor.

In some embodiments of the method, the flow rate of cells from one end of each segment to the other end is determined by the cell division rate. In some embodiments, cells divide once during the time period in which they are transported from one end of the segment to the other end of the segment. In some embodiments, the flow of liquid medium through each segment is laminar flow.

Also provided herein are methods for mass production of cells, comprising: introducing cells into a production bioreactor, wherein the production bioreactor comprises a collection of mini-modules of double helical tetrahedral shapes or structures, wherein the mini-modules are arranged in layers within a macrostructure; flowing a liquid medium into the production bioreactor; and supplying a gas composition into the production bioreactor; wherein the cells are transported between the mini-modules; and wherein the cells are transported from an inlet end of the macrostructure to an outlet end of the macrostructure. In some embodiments of the method, the cells divide once on average during transport from one layer of a mini-module to the next layer of a mini-module. In some embodiments, the amount of liquid medium flowing to each level of the mini-module maintains substantially the same cell density in each level. In some embodiments, the velocity of the liquid medium flowing through the production reactor exceeds the free-fall velocity of the cells.

In some embodiments of the methods herein, cells are introduced from a cell chip or a sandbox module into a bioreactor. In some embodiments, a portion of the cells is collected from an output end of a macrostructure. In some embodiments, the cells produce at least one bioproduct, and wherein the bioproduct is collected from an outlet end of the macrostructure, for example, a small molecule, protein, antibody, large macromolecule, metabolite, or a combination thereof produced by the cells.

Provided herein are methods for customized cell production, comprising: introducing cells of a selected type into a cell chip; growing the cells in the cell chip; transferring the cells from the cell chip module to a sandbox bioreactor; and optimizing at least one parameter of a first cell environment in the sandbox bioreactor to produce a second cell environment. In some embodiments, the method further comprises growing a sample of cells of the selected type in a production bioreactor having the second cell environment. In some embodiments, cells or portions thereof are collected from the production bioreactor. In some embodiments, the cells used in the method are CAR-T cells, stem cells, or differentiated cells, or a combination thereof. In some embodiments, the cells produce at least one bioproduct and wherein the bioproduct is collected from the production bioreactor.

Provided herein are methods for growing cells, comprising growing cells in a cell chip module as described herein. In some embodiments, the method further comprises placing the cells in a storage mode. Provided herein are methods for optimizing cell environmental conditions, comprising growing cells in a sandbox bioreactor as described herein and optimizing at least one parameter of the cell environment. Also provided herein are methods for growing cells, comprising growing cells in a production bioreactor as described herein. In some embodiments, the method is a method for high-scale cell production. In some embodiments of the methods for growing cells and producing cells, the cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, animal cells, avian cells, mammalian cells, human cells, and genetically modified cells. In some embodiments, the systems, devices, and methods described herein can be used in zero gravity or in microgravity conditions to grow cells in zero gravity or microgravity conditions.

Those skilled in the art will readily appreciate additional aspects and advantages of the present invention from the following detailed description, wherein only illustrative embodiments of the present invention are shown and described. As will be appreciated, the present invention is capable of other and different embodiments, and that its several details are capable of modification in various significant respects, all without departing from the present invention. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature and not restrictive. Incorporated by Reference

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was expressly and individually indicated to be incorporated by reference. To the extent the disclosure contained in this specification is inconsistent with the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such conflicting material.

CROSS REFERENCE This application claims the benefit of U.S. Provisional Patent Application No. 62/743,974 filed on October 10, 2018, the entirety of which is incorporated herein by reference.

Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art may conceive of numerous variations, changes, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

Provided herein are systems, components, and methods for producing and maintaining cells and for producing and isolating cells and products made from cells. The systems, components, and methods herein provide flexibility for customized production for different cell types, types of cell environments, and types of molecules produced. The systems, components, and methods also provide scale flexibility. For example, the systems, components, and methods described herein can provide production scale-up without changing or significantly changing laboratory-scale growth conditions.

Included herein are one or more bioreactors for growing cells in systems and assemblies. The bioreactors are on a microbial reactor scale, allowing the system to be constructed as a benchtop bioreactor with the ability to grow and produce small and large quantities of cells and/or cell products. Such systems and methods of use are advantageous in their scalability, flexibility, and resource conservation.

As used herein, the term "cell chip" or "cell chip module" generally refers to a device suitable for growing, culturing and/or storing cells, which may include one or more channels or other openings for inputting cells, for providing liquid culture medium and other cell environmental factors, and, optionally, one or more structures for capturing, containing or directing the flow of cells within the growth/culture environment of the cell chip or a subsection thereof.

As used herein, the term "sandbox bioreactor" generally refers to a device used to grow cells and allows the effects of one or more cell environmental conditions to be tested in a repetitive manner on one or more types of cells. A sandbox reactor may include a design such that the cell environment therein and the effects of the cell environment on the cells are relevant to the cell environment and effects for scale-up and production of cells and bioproducts. A sandbox reactor may include a single test environment or may be composed of multiple segments in which one or more environmental conditions may be tested.

As used herein, the term "production bioreactor" or "bioreactor" generally refers to a bioreactor device suitable for large-scale production of cells and/or products produced by cells. A production bioreactor may include one or more channels or other openings for inputting cells, for providing liquid culture medium, gas composition and other cellular environmental factors, and one or more channels for harvesting cells and/or products produced by cells.

As used herein, the term "media preparer" generally refers to a component or device used to mix the components of a culture medium used to grow cells.

As used herein, the term "trap" generally refers to a structure used to capture, contain, and/or direct the flow of cells within a solid area. Traps may include, but are not limited to, gate traps, overflow traps, suction traps, and porous membrane traps (such as, but not limited to, those described herein). In some embodiments, the size of the trap is determined by the cell size, volume, and/or diameter, such that the trap slows, stops, or prevents the movement of cells from one solid space to another. In some embodiments, the ability of the capture device to slow, arrest, or prevent cell movement can be reduced or overcome as the number or density of cells increases or as the flow rate of cells or liquid medium increases.

As used herein, the term "mini-module" generally refers to fragments of a production bioreactor that can be interconnected and assembled into a larger structure (e.g., a macrostructure or macroshape) to constitute at least a portion or the entirety of the production bioreactor.

As used herein, the term "helical icosahedron" generally refers to a connected periodic minimal surface that contains no straight lines. This surface can have a mathematically infinite number of connections. In some examples, the helical icosahedron is a unique non-trivial embedded component of a related series of Schwartz P and D surfaces with a correlation angle of about 38.01°. The helical icosahedron can be configured as a single helical icosahedron or a double helical icosahedron. The double helical icosahedron can be oriented and configured for specific applications in microfluidic devices. The double helical icosahedron can be configured by balancing geometric states (such as double helical icosahedron crystal structure and spatial grouping) that are related to fluid dynamic performance observed in mini-modules and macrostructures (e.g., macroscopic shapes). The helical icosahedron or the double helical icosahedron can be implemented in various crystal structures.

Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first value in a series of two or more numerical values, the term "at least," "greater than," or "greater than or equal to" applies to each of the numerical values in the series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

Whenever the term "not more than,""lessthan," or "less than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "not more than,""lessthan," or "less than or equal to" applies to each of those numerical values in the series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1. Bioreactor Module

In one embodiment, the present invention provides a system and method for modular and interconnected bioreactor components for producing cells. The cells can be bacterial cells, fungal cells, yeast cells, eukaryotic cells, plant cells, or algae cells. The cells can be recombinant cells. The modular system can include a first module, a second module, and a third module. The first module can be a cell chip configured to accommodate a plurality of cells. The second module can be in fluid communication with the first module and can be a sandbox reactor. The sandbox reactor can be configured to (i) interface with the cell chip, (ii) direct a subset of cells from the plurality of cells to different sections of the sandbox, and (iii) repeatedly generate a set of growth conditions for the plurality of cells. The different sections can be configured individually. The third module can be fluidly connected to the first module and the second module. The third module can include a bioreactor, which is configured to (i) interface with the second module, (ii) receive the subset of cells, and (iii) generate a copy of the subset of cells under the set of growth conditions. In some embodiments, the third module can include a bioreactor and can be fluidly connected to the cell chip module.

The systems, components and methods herein are modular and interconnectable. In some embodiments herein, the system is composed of one or more modules, wherein each module comprises at least one bioreactor. In some embodiments, the system contains at least one, two, three or more modules. In some embodiments, the system comprises more than three modules. Each module is configured for laminar flow of liquids (including culture medium and/or solvents) and further in some embodiments for unidirectional flow of cells. In some embodiments, one or more modules are configured for transitional flow or turbulent flow of liquids (e.g., culture medium and/or solvents).

In some embodiments, the system comprises at least 1, 2, 3 or more modules interconnected. In some embodiments, the modules include one or more of a cell chip module, a sandbox bioreactor module, and a production bioreactor module. In some embodiments, the cell chip module is interconnected (e.g., fluidly connected) to the sandbox bioreactor module so that the cell chip supplies cells to initially inoculate the sandbox reactor. In some embodiments, the cell chip module is interconnected (e.g., fluidly connected) to the production bioreactor so that the cell chip supplies cells to initially inoculate the production bioreactor. In some embodiments, the sandbox bioreactor is interconnected (e.g., fluidly connected) to the production bioreactor and cells from the sandbox bioreactor flow into the production bioreactor for scale-up. In some embodiments, the system connects the cell chip to the sandbox bioreactor and the production bioreactor in a series configuration. Alternatively or in addition, the system connects the cell chip to the sandbox bioreactor and the production bioreactor in a parallel configuration. Cells can be initially grown in the cell chip, tested in the sandbox bioreactor for optimal environmental conditions and scaled up in the production bioreactor.

In some embodiments, the system includes a module as a cell holding and storage chip (also referred to herein as a cell chip) into which one or more cells are input and the one or more cells are proliferated through cell growth and division. The cell chip may be a consumable (e.g., used once or a few times and discarded). The cell chip may have one or more cell lines. The cell chip may include an input channel for flow in liquid culture medium and an output channel for used culture medium and, if necessary, cell flow out of the cell chip. The cell chip may have at least 1, 2, 3, 4, 5, 6, 8, 10 or more input channels. The cell chip may have at least about 1, 2, 3, 4, 5, 6, 8, 10 or more output channels. The cell chip may have the same number of input and output channels or may have different numbers of input and output channels. Each input channel may flow a single component (e.g., a single liquid medium) or each input channel may flow different components (e.g., different types of liquid medium). The output channels may flow excess or excess medium and cells. Alternatively or in addition, cells may be separated from the medium flow and exported from the cell chip by one or more output channels. The cell chip may include at least one mechanism for capturing cells within a module.

In some embodiments, the mechanism for capturing cells is a gate trap that collects cells in a trap as the cells flow into the trap along with the laminar flow of the liquid culture medium in the cell chip. The gate trap can prevent cells from flowing freely in the chip, confine the cells to a shielded environment, and not be affected by flow inertial forces, while enabling proper access to nutrients and gases. The gate trap may have an inoculation port to enable cells to be inoculated in the internal space of the gate trap. The gate trap may have one or more flow protectors that are shaped so that the internal cells are not affected by flow inertial forces. Exemplary shapes or structures that can be used as the one or more flow protectors include triangles, squares, pentagons, hexagons, circles, and combinations thereof. The flow protectors can be interrupted by openings to allow access to gases and nutrients, and to allow cells to escape from the gate trap in a controlled manner. In some embodiments, the protector opening size is larger than the diameter of the cells within the flow protector. In some cases, the opening size is twice the size of the cells within the flow protector. In some cases, the opening size can be at least 2 times, 3 times, 4 times, 5 times, 7 times, or 10 times larger than the diameter of the cells within the flow protector. In some embodiments, the openings are evenly distributed throughout the shape or structure of the flow protector shape. In some embodiments, the openings are configured according to the expected flow direction and speed. In some embodiments, the openings may be more sufficient on the second half of the flow protector shape, counting in the general direction of flow. In some embodiments, the diameter of the gate catcher is from about 20 microns to about 1000 microns. In some embodiments, the diameter of the gate catcher is about greater than or equal to 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns or more. In some embodiments, the diameter of the gate catcher is about 100 microns, 200 microns, 300 microns, 400 microns, 500 microns, 600 microns, 700 microns, 800 microns, 900 microns, 1000 microns or more. In some embodiments, the diameter of the gate catcher is less than or equal to about 1000 microns, 900 microns, 800 microns, 700 microns, 600 microns, 500 microns, 400 microns, 300 microns, 200 microns, 100 microns, 90 microns, 80 microns, 70 microns, 60 microns, 50 microns, 40 microns, 30 microns, or less.

In some embodiments, the mechanism for trapping cells is an overflow type trap. The overflow type trap(s) can prevent cells from flowing freely within the chip by physically preventing cells from escaping and enforcing their restrictions using inertial flow forces, thereby confining cells to a shielded environment while being able to access nutrients and gases. In some embodiments, the overflow trap flow protector includes one or more canalized walls sequentially configured to physically confine cells within the trap. In some embodiments, the channelized walls have openings therebetween. In some embodiments, the openings are smaller than the internal cell diameter. In some embodiments, the channelized walls do not have any openings therebetween. In some embodiments, there is an opening between the top of the chip and the height of the channeling box wall. In some embodiments, the opening between the height of the channeling box wall and the top is smaller than the diameter of the cells in the trap. When the cell volume exceeds the volume capacity of the gate trap, the cells flow out of the trap. In some embodiments, the diameter of the overflow trap is about 50 microns to about 2000 microns. In some embodiments, the diameter of the overflow trap is greater than or equal to about 180 microns or 210 microns. In some embodiments, the diameter of the overflow trap is less than or equal to about 210 microns or 180 microns. In some embodiments, the diameter of the overflow catcher is greater than or equal to about 50 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, 600 microns, 800 microns, 1000 microns, 1200 microns, 1500 microns, 2000 microns or more. In some embodiments, the diameter of the overflow catcher is less than or equal to about 2000 microns, 1500 microns, 1200 microns, 1000 microns, 800 microns, 600 microns, 500 microns, 450 microns, 400 microns, 350 microns, 300 microns, 250 microns, 200 microns, 150 microns, 100 microns, 50 microns or less.

In some embodiments, the cell trap comprises a suction trap. The suction trap can be designed by vertically separating a liquid medium region from a cell growth region with a porous membrane. The suction trap can be designed to have one or more arms where cells are input and where cells grow and divide. The liquid medium travels through the chip and can cross the porous membrane to the lower region, while the cells remain in the upper region due to the pore size being smaller than the cell size. The movement of the liquid medium from the upper level to the lower level creates a suction force that keeps the cells within the one or more arms. When the cells divide to a certain number or density, the suction force no longer maintains all cells in the arms, and the cells can flow out of the arms on the upper level and toward the output port of the cell chip. The suction trap may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more arms. In some embodiments, the porous membrane used may vary depending on the cell type. In some embodiments, the porous membrane may have a pore size smaller than the cell diameter. In some embodiments, the pore size of the porous membrane may be greater than or equal to 0.22 microns, 1 micron, 3 microns, 5 microns, 7 microns or more. In some embodiments, the pore size is less than or equal to about 7 microns, 5 microns, 3 microns, 1 micron, 0.22 microns or less. In some embodiments, the porous membrane used has discrete pores of a selected specific pore size; this can be achieved by implementing a track etching technique. In some embodiments, the pores of the porous membrane are randomly distributed. Alternatively or in addition, the pores can be configured or patterned. In some embodiments, the material used for the porous membrane is a high-quality polycarbonate membrane. In some embodiments, the material used for the porous membrane is polysulfone, polyethylene, polytetrafluoroethylene, polypropylene, nitrocellulose, nanocellulose, nylon, ceramic foam or carbon nanotubes.

In some embodiments, the mechanism for trapping cells in a cell chip comprises a combination of trap types, including gate traps, suction traps, and overflow traps. In some embodiments, the trap types are connected in series, so that when cells are released from one type of trap, the cells flow to and are trapped in another type of trap. Alternatively or in addition, the traps may be configured in a parallel configuration. In some embodiments, the traps are configured in both series and parallel configurations. The number and configuration of the traps can be customized for the cell type as well as the size and volume of the cell chip and the liquid flow rate in the cell chip. In some embodiments, the cell chip individually accommodates greater than or equal to 1, 2, 3, 4, 5, 10, 20, 50, 100 or more gate traps, or accommodates such gate traps together with one or more overflow traps or together with one or more suction traps. In some embodiments, the cell chip individually accommodates greater than or equal to about 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 300, 400, 500, 1000 or more overflow traps, or accommodates such overflow traps together with one or more gate traps or one or more suction traps.

In some embodiments, the mechanism for trapping cells is a porous membrane trap. This type of trap has a liquid medium flow on the upper side of the porous membrane and cells are introduced and grown in a chamber below the porous membrane. As medium is perfused into the cell chamber and as the cell density increases and the cells grow closer to the membrane, the cells are drawn through the pores of the membrane into the upper medium channel where they can then move with the liquid medium flow, thereby leaving the trap. The pore size of the membrane in the trap can be customized for the cell type and size. In some embodiments, the pore size is from about 0.22 microns to about 5 microns. In some embodiments, the pore size is greater than or equal to about 0.22 microns, 1 micron, 3 microns, 5 microns, 7 microns, or more. In some embodiments, the pore size is less than or equal to about 7 microns, 5 microns, 3 microns, 1 micron, 0.22 microns, or less. The size and volume of the chamber below the diaphragm can be customized for the cell type and size. The number of cell chambers can be customized for any processing volume. In some embodiments, the number of cell chambers is greater than or equal to 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1000, or more.

In some embodiments, after the cells are grown in the cell chip, the cell chip stores the cells captured therein for later use. The cells can be stored by various methods, for example, including freezing, drying, lyophilization, or storage in a liquid culture medium or a liquid culture medium modified to enable cell storage at room temperature or at other temperatures.

In some embodiments, the system includes a sandbox bioreactor. The sandbox reactor may be a consumable (e.g., used once or several times and discarded). Alternatively or in addition, the sandbox reactor may not be a consumable. The sandbox bioreactor module is composed of a series of fragments, each of which has at least one microchannel suitable for transferring cells from one end of the microchannel to the other end of the microchannel. In some embodiments, the fragment has two or more microchannels that act in parallel with each other, so that cells in one microchannel may not be transferred to another microchannel in the same fragment. In some embodiments, the fragment has greater than or equal to 2, 4, 8, 16, 32, 64, 128 or ²ⁿ microchannels in parallel. The length and diameter of the microchannel can be tailored to the cell type, cell size, and the rate of transport of cells and liquid medium in the microchannel. In some embodiments, the channel can have a diameter greater than or equal to about 20 microns, 50 microns, 70 microns, 120 microns, 200 microns, 600 microns, 1500 microns, 2000 microns and a length of about 1 mm, 2 mm, 4 mm, 10 mm, 22 mm, 40 mm, 120 mm, 200 mm or more. In some embodiments, the length of the microchannel is tailored to the cell division rate of cells transported through the microchannel, so that the cell divides once during the transit time from entering the microchannel to leaving the microchannel. The diameter of each microchannel can be customized for cell size, mixing rate of transported fluids, gas exchange rate, or a combination thereof.

In some embodiments, a segment of a sandbox bioreactor includes two or more sections. One section may include a mixing module and another section may include a growth chamber. The mixing module and the growth chamber may be in a series or parallel configuration. In an example, the mixing module and the growth chamber are in a series configuration. The mixing module may be fluidically connected to greater than or equal to 1, 2, 4, 6, 8, 10 or more input channels. The input channels may include channels for flowing culture media, gases and/or cells. In an example, the mixing module is fluidically connected to two input channels so that one channel provides culture media and the other channel provides cells to the mixing module. Culture media and cells can be mixed in the mixing module. The mixing module may include straight channels or may include channels with serpentine or curved geometric structures. The channel of the mixing module may have a diameter greater than or equal to about 20, 50, 70, 120, 200, 600, 1500, or 2000 microns and a length of about 1, 2, 4, 10, 22, 40, 120, or 200 mm. The mixing module may provide mixed cells and culture medium to the growth chamber. The growth chamber may include an expansion zone that increases the diameter of the fragment. The growth chamber may have a diameter greater than or equal to 20, 50, 70, 120, 200, 600, 1500, or 2000 microns and a length of about 1, 2, 4, 10, 22, 40, 120, or 200 mm.

In some embodiments, the sandbox bioreactor has two or more segments placed in series with each other so that cells from the microchannel of one segment flow into the microchannel of the next segment. In some embodiments, the microchannel from the first segment is interconnected to two microchannels in the second segment placed in series with the first segment. The length of the microchannel of the first segment is customized so that the cell divides at most once during the transit time from entering the first microchannel to leaving the first microchannel, and so that the two cells generated from this division are separated and flow into the two microchannels of the second segment respectively. The two microchannels of the second segment can be configured in a parallel configuration. In some embodiments, the microchannels of the second segment are interconnected in a manner similar to the microchannels of the next segment in series. In some embodiments, the cells do not divide during their transit through each microchannel. In other embodiments, cells can divide greater than or equal to 1, 2, 3, 4, 5 or more times during a time period of the length of their transport through the microchannel. The segments can be placed in series in greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more segments, each segment containing one or more microchannels. In the case where the microchannels are interconnected from one segment to the next and dividing cells are separated when cells are transported from the microchannels of one segment to the next, the number of microchannels can be increased up to ^2n- fold, where n = 1, 2, 3, 4, 5, 6 or greater than 6.

Sandbox bioreactors can allow the effects of changing environmental conditions on cells to be tested and measured. Changing environmental conditions can include changes in medium composition, pH, temperature, gas composition, gas exchange, cell density, cell flow, and/or flow rate of liquid medium. In some embodiments, the sandbox bioreactor has an inlet to a first segment through which liquid medium is provided and flows through the first segment to downstream segments connected in series and/or parallel. The composition and/or flow rate of the medium can vary over time or can remain constant,

The sandbox bioreactor may include one or more sensors and/or one or more sample collection devices. The sensors and/or sample collection devices can be used to monitor cellular responses to environmental changes. In some embodiments, one or more sensors are online sensors. In some embodiments, the sensors are offline and receive samples of cells, culture media, or a combination thereof. The sensors can measure biological, physical, and/or chemical parameters. Exemplary parameters are pH, cell division rate, cell growth rate, cell density, temperature, optical density, gas composition, and/or gas exchange rate. Exemplary parameters also include profiling, single species identification, and quantification of one or more of cellular metabolites, proteins, nucleic acids, lipids, small molecules or biomolecules, or combinations thereof. In some embodiments, the sensor measures cellular stress responses and cellular metabolites as a response to environmental changes.

The sandbox reactor may further include one or more controllers. The controllers may control the cell environment in one or more sections of the sandbox bioreactor. In some embodiments, the controller communicates with one or more sensors related to the cells, cell environment, or a combination thereof within the one or more sections of the sandbox bioreactor and/or receives information from the one or more sensors. The controller may change the cell environment in response to information from the one or more sensors. Example changes include liquid medium flow rate, liquid medium component concentration, pH, temperature, gas concentration, gas content, and cell density.

In some embodiments, the system includes a production bioreactor module. The production bioreactor provides an environment for the scaled-up growth and production of cells and/or bioproducts from the cells. The production bioreactor provides a 3-D structure comprising a plurality of mini-modules. The production bioreactor may include greater than or equal to 1, 2, 4, 6, 8, 10 or more mini-modules. The mini-modules create a series of channels and chambers for the growth and movement of cells and for the flow of liquid culture media, gases and bioproducts. The mini-modules of the production bioreactor may include a number of shapes, such as a double helical tetrahedron, a modified double helical tetrahedron, or any shape that can be described as a triple periodic minimal surface (TPMS). This type of surface forms a grid system that can be grown periodically on all three axes (X, Y, Z). A TPMS may have no self-intersection points and divide a given volume into two (or more) independent sub-volumes. Self-intersection points may include a surface where a single normal vector at each point defines the surface. If the surface divides a volume in which it is circumscribed into two independent and congruent sub-volumes, then the surface is called a balanced surface. The TPMS is described in terms of a basic patch or asymmetric unit from which the entire surface can be built by its symmetric elements. The mini-modules can be fluidly connected to each other (e.g., interconnected) so that gases, culture media and/or byproducts can flow from one mini-module to another.

In some embodiments, a mini-module for a production bioreactor may comprise a double helical icosahedron or a modified double helical icosahedron shape. A double helical icosahedron (DG) may comprise two helical icosahedrons and may comprise two inter-occurring non-overlapping domains. A modified double helical icosahedron (DG) may comprise two inter-occurring non-overlapping domains that may be bounded by two constant mean curvature (CMC) surfaces separated by a matrix phase. A modified double helical icosahedron structure may comprise minimal modifications to the connectors of an unmodified double helical icosahedron in order to adapt the structure to a given macrostructure or function. Modifications may include blocking portions of such connections or intersections (e.g., "mouths"), modifying the diameter of one or both phase channels of the structure, or completely or partially eliminating any phase channels present in the DG structure. The DG or modified DG may include a first helical icosahedral structure entangled with a second helical icosahedral structure. The two channels may be separated by a porous membrane (matrix phase). The matrix phase may diffuse gas molecules in a manner that may be based at least in part on a specific pressure and gas composition. When the radius of the liquid component microchannel is equal to the radius of the gaseous component microchannel, the matrix phase surface may be equal to the sum of these radii. When multiple DGs are interconnected (e.g., fitted together), two CMC surfaces create two continuous channels. These two channels create two non-overlapping channels for the flow of liquid medium and/or gas. The porous membrane can provide a surface on which a specific cell type can adhere and grow. In some embodiments, one channel provides liquid medium throughout the production bioreactor. In some embodiments, both channels provide liquid. In some embodiments, one channel provides liquid medium and the other channel provides gas to the production bioreactor. In some embodiments, the diameter of the microchannel of the mini-module can be changed as needed for specific cell types, production requirements, and the like. In some embodiments, the mini-module can have a regular cubic coating structure with an edge having a length "L". L can be related to the sweeping diameter. In some embodiments, L is equal to two-thirds of the swept diameter of the microchannel, times the square root of 2, times the square root of 3. If the radii of both the liquid component and the gaseous component are the same within a minimodule, the total surface and volume of the microchannels corresponding to the liquid component can be equal to the corresponding dimensions of the gaseous component. In some embodiments, the radii of the components can be different. In some embodiments, when the two radii are the same, the microchannel radius cannot be larger than 0.7 times the swept radius. The shortest distance between two minimodules on two different sides is equal to the swept radius times the square root of 2, minus the sum of the radii of the channels of each component.

In some embodiments, the area of a first channel of the DG is equivalent to the area of a second channel in the DG, and the area of the matrix phase is the sum of the area of the first channel and the area of the second channel.

The distance between the matrix phase separating the channels from the center of each channel is constant. The rate at which the medium and gas flow through the production module can be determined by selecting the cell type and cell density and the pressure conditions to be generated on the cells. The rate at which the gas diffuses through the matrix into the liquid medium is determined by the gas composition and gas pressure in the gas channels formed by the structure, as well as the thickness of the diaphragm and the materials selected to make the channels and surrounding areas. The gas flow rate and working pressure can be related to the culture cell density. In some embodiments, the gas flow rate can be equal to the volume of the gaseous component per minute. In some embodiments, the gas flow rate is greater than or equal to about 2, 3, 5, or 10 times the volume of the gaseous component per minute. In some embodiments, the operating pressure can be varied from about 1 atmosphere (atm) to 5 atm. In some embodiments, the operating pressure is greater than or equal to 1 atm, 2 atm, 3 atm, 4 atm, 5 atm, or more.

The advantage of DG is that it reduces gravity forces that in other structures can provide uneven media exposure and provide gas exchange. The shape creates three-dimensional (3-D) laminar flow forces that average out variations in the distance of any one cell to the structure wall to provide more constant and more uniform exposure among the cell population. In addition, the DG shape avoids stagnant areas of liquid or gas where flow may not occur or may be interrupted. This allows the use of higher throughputs at lower speeds through the bioreactor and results in lower shear pressures on the cells. In some embodiments, the average velocity may be greater than or equal to about 1 μm/sec, 3 μm/sec, 5 μm/sec, 10 μm/sec, 15 μm/sec, 20 μm/sec, 50 μm/sec, 100 μm/sec, 200 μm/sec, or more. The DG structure provides for better diffusion of media and gases compared to other bioreactor systems. In some embodiments, the velocity of liquid media flowing through channels within the DG is greater than the free-fall velocity of cells flowing through the same channels.

DG structures offer increased surface area in many shape options, and this increased surface area provides surface area for cell growth and improved liquid medium flow, mixing, and gas exchange. When L equals L1, the surface of each component can be described as Y = 3258.6.XE(-1), where Y is square millimeters/microliters and X equals the radius defined by L1.

The mini-module DG structures are assembled together into a macrostructure or macroshape that constitutes a production bioreactor. In some embodiments, the macrostructure is a pyramid. In some embodiments, the macrostructure is a hollow pyramid, a thin pyramid, a checkerboard configuration, or a log. The number of macrostructures and mini-modules within a production bioreactor can be customized for the cell division rate of the cells to be grown, as well as to regulate the liquid medium, gas exchange, and the speed of cell movement through the bioreactor. Each macrostructure can provide different possibilities for interaction with the cells, and the macrostructure can be selected in view of the specific process that the production bioreactor is expected to stimulate. Pyramid and hollow pyramid macrostructures achieve a suitable environment for growth while maintaining a constant speed and cell density. More sensitive strains may require more intervention over time, in which case the hollow pyramids can provide that ability. Lamellar pyramids achieve a suitable environment for growth and development by keeping both speed and density constant while providing full access to each cell at every time point, thereby achieving direct intervention and treatment. Checkerboard and log configurations can also provide full access to each cell at every time point in the procedure while allowing control of uniform speed and density. In some embodiments, cells are input at the top of the macrostructure and at the cell collection device at the base of the macrostructure.

The DG mini-modules are configured as a macrostructure to provide a mechanism for determining and optimizing the flow of liquid medium and gas in the bioreactor. In some embodiments, the macrostructure includes several layers or levels of mini-modules. In some embodiments, the mini-modules are configured into several layers or levels and the speed of the liquid medium in each layer is substantially the same. Alternatively or in addition, the speed of the liquid medium in each level or layer can be changed. For example, the speed of the liquid medium can increase or decrease between levels or layers. The speed of the liquid medium can change between mini-modules or can be substantially the same between mini-modules. In some embodiments, the production bioreactor further includes a liquid medium input device. The liquid medium device can be structured to provide liquid medium to each level of mini-modules within the macrostructure. In some embodiments, the volume of liquid medium provided to each level maintains a substantially constant cell density in each level. The microchannel radius can be associated with the cell radius, cell density, or other parameters (e.g., filamentary configuration, chain configuration, etc.). In some embodiments, the cell density can be changed from 1 x 10 ⁶ cells/ml to 1 x 10 ¹² cells/ml. In some embodiments, the rate at which liquid medium passes through each mini-module is determined by the cell division rate, such that the time it takes for a cell to pass through a single mini-module or a layer of mini-modules is substantially the same as or proportional to the cell division rate, such that a cell divides greater than or equal to 1, 2, 3, 4, 5, or more than 5 times during transport. In some embodiments, a first layer has x volume of liquid medium such that at a given number of cells, the density is X, and a second layer has 2x volume of liquid medium, and over a sustained period of time cells are transferred from the first layer to the second layer, the number of cells doubles (e.g., each cell divides once on average) such that the density in the second layer remains X (i.e., a constant cell density between layers).

Additional optimization can be achieved by determining the expected number of cells at the base of the macrostructure (the end of the macrostructure where cells and/or bioproducts arrive before exiting the structure through the output to a collection vessel). Expected cell numbers can also be determined for different layers of the macrostructure. Based on the expected cell numbers at the base and different layers, the flow of gas and liquid media can be adjusted for each layer to compensate for increased gas and liquid media requirements as the cell number increases through cell division, cell migration, and cell accumulation as the cells travel through the bioreactor toward the base of the structure. Liquid Media Supply

In some embodiments, the system includes one or more components to supply liquid medium to one or more modules. The components may include one or more of a medium preparer, an electroporator or other sterilization device, a container, a pump, a bubble sensor, and a bubble trap. The medium preparer generates liquid medium for one or more modules by appropriately mixing the components of the medium with water to grow cells in the module. The electroporator can be interconnected to the medium generator to clean the medium and provide a sterile starting medium to supply to one or more modules to grow cells. A bubble sensor and bubble trap may be included to detect and remove any bubbles in the liquid medium introduced during medium generation or cleaning.

The system may also include one or more containers for holding a reserve medium before supplying the reserve medium to the modules. In some embodiments, the system includes at least 2, 3, 4, 6, 8, 10 or more containers. The containers can be loaded asynchronously so that one container is loaded while another container that is fully loaded is used to supply liquid medium to one or more modules. Separating the containers in this manner is particularly advantageous for isolating the cell growth modules of the system from any connection to an electrical current. The loaded containers are exposed to an electrical current that can flow from an upstream component such as an electroporator. The loaded container is isolated from the current flow so that it cannot transmit current to downstream components and modules. In some embodiments, the size of the container can be related to the throughput of the production bioreactor within the division time of the cells selected for the process. In some embodiments, multiple containers can be mounted in parallel with each other and decoupled from each other. In some embodiments, multiple containers can be mounted in series.

The liquid medium supply assembly may also include one or more sensors. These sensors can measure parameters including pH and temperature of the medium. The sensor may be an online sensor or may be connected to a sampling device that intermittently samples the medium from one or more components of the liquid medium supply. The supply system may provide liquid medium at a rate range depending on the purpose, scale, and operation of the system. In some embodiments, the liquid medium supply may provide from about 100 microliters to about 1000 liters per hour to one or more of the downstream modules. In some embodiments, the liquid medium supply may provide from about 0.5 liters to 1000 liters per hour to one or more of the downstream modules. In some embodiments, the liquid medium supplier provides from about 0.5 liters to 5 liters per hour to one or more downstream modules. In some embodiments, the liquid medium supplier provides from about 10 liters to 80 liters per hour to one or more downstream modules. In some embodiments, the liquid medium supplier provides from about 100 liters to 1000 liters per hour to one or more downstream modules.

The liquid medium supply assembly may include one or more pumps for flowing the medium from the accumulator or medium preparer to downstream components (such as cell chips, sandbox bioreactors, or production bioreactors). The system may include greater than or equal to 1, 2, 3, 4, 6, 8, 10 or more pumps. The pumps may be pumps of the same type or may be pumps of different types. Exemplary pumps include syringe pumps, peristaltic pumps, and pressure pumps. The liquid medium supply system is configured to provide unidirectional flow to downstream components. In some embodiments, the pump is a syringe pump for supplying medium to the cell chip. In some embodiments, the pump is a syringe pump for supplying medium to the sandbox bioreactor. In some embodiments, the pump is a peristaltic pump for supplying culture medium to a production bioreactor. In some embodiments, the system comprises three pumps, namely, two syringe pumps for supplying the cell chip and sandbox reactor and a peristaltic pump for supplying the production bioreactor. The pumps may operate synchronously or individually. In some embodiments, all three pumps operate synchronously. One or more pumps supply culture medium to downstream modules with a high degree of volume and rate accuracy. In some embodiments, the accuracy is within 1 nanoliter, 2 nanoliters, 3 nanoliters, 4 nanoliters, or 5 nanoliters. Gas Supplies and Compositions

The systems herein are compatible for use with cells that require specific gas compositions, such as cells that require oxygen to grow and survive. Materials used to construct bioreactor modules may include glass, acrylic, collagen, polydimethylsiloxane (PDMS), poly(ethylene glycol) (PEGDA), poly(D,L-lactide), and other biocompatible polymers that allow oxygenation of the culture medium. In some embodiments, the system includes a controller that controls the diffusion of oxygen and other gas solutions in one or more modules. The gas solutions are prepared from pure component gases, such as from gas tanks or other supplies, to create mixtures or pure gas solutions at various concentrations and flow rates. Alternatively or in addition, the gas mixture may be provided by a purified air mixture. Gas solutions can be used to provide an aerated environment and control pH, as well as provide carbon, nitrogen, phosphorus, and sulfur to the liquid phase. In some embodiments, the system has more than one gas controller or mechanism so that different gas solutions can be provided to different modules within the system. Sensors and Environmental Monitoring

In some embodiments, one or more bioreactor modules are interconnected with one or more sensors that monitor one or more characteristics of the environment. The sensors can be placed at specific points within the module or system. Measurements are taken within a specific time frame and at specific time points so that the sensors monitor specific cell populations. In some embodiments, one or more sensors track the characteristics of a specific cell population as it moves through the bioreactor module. The sensors can trigger specific treatments in response to the measurements to optimize the conditions for the specific cell population. Exemplary measurement and optimization of characteristics include pH, diluted oxygen, total gas composition, dilution of other gases, temperature, cell density, glycoprofile, transcriptomics and target transcript monitoring, metabolomics, and proteomics. In some embodiments, characteristics are measured at at least 1, 2, 3, 4, 6, 8, 10 or more time points. In some embodiments, characteristics are measured for a specific cell population and can be timed to monitor the specific cell population as it moves through the modules of the bioreactor and between different modules.

In some embodiments, at least one sensor measures a parameter, such as a biological parameter, a physical parameter, or a chemical parameter, of cells within, traveling through, entering, and/or exiting a bioreactor module. In some embodiments, at least one sensor measures a parameter, such as a biological parameter, a physical parameter, or a chemical parameter, of the cell environment within the bioreactor module. Exemplary biological parameters include cell division rate, cell growth rate, cell stress response, cell protein content, cell carbohydrate content, cell lipid content, and cell nucleic acid content. Exemplary physical parameters include cell size, cell density, cell flow rate, liquid medium flow rate, mixing rate, turbidity, temperature, and pressure. Exemplary chemical parameters include pH, liquid medium composition, concentrations of individual liquid medium components, gas composition, gas concentration, and dissolved gas composition.

In some embodiments, the system or module is in communication with a camera device. The camera monitors the output of at least one bioreactor module. The camera can capture biochemical, physical, or chemical characteristics of the bioreactor output. In some embodiments, the camera device captures information such as cell count, cell velocity, and cell density. In some embodiments, the camera is a reverse spectrum camera that captures information across a range of wavelengths. In some embodiments, the camera device captures information from the output of two or more bioreactor modules. Methods of Use

In some embodiments herein, one or more modules are used to produce cells. FIG. 8 shows an exemplary method of producing cells using one or more modules. Cells can be collected through an output channel of a module. In some embodiments, cells are collected within the module and stored in the module, such as storing the cells in a cell chip module. In some embodiments, one or more modules are used to produce biological products from cells, such as small molecules, proteins, antibodies, metabolites, or other products produced by cells grown in the module. The biological products can be collected through an output channel of a module and separated from the growing cells, such as by diffusion through a porous membrane or by filtration. In some embodiments, the bioproduct is inside the cell. To harvest the bioproduct, the cells are harvested, lysed, and the bioproduct may then be further purified. In some embodiments, the bioproduct is secreted from the cell and may be collected without harvesting or lysing the cell.

The bioreactor modules and systems herein can be used to produce specific cell types and have the flexibility to grow a variety of cell types. In some embodiments, a cell chip module is used to provide a specific type of cell to a sandbox bioreactor, to a production bioreactor, or to a sandbox module in series with a production module. The cell chip module allows for customized cell production and environmental optimization.

The cell chip can be adapted to produce stem cells and other types of cell therapies, including autologous and allogeneic production. In some embodiments, expansion, gene delivery or activation of T cells can be performed for personalized chimeric antigen receptor T cell (CAR-T) therapy. In some embodiments, the stem cells can be undifferentiated, growing and/or differentiated.

In some embodiments, the cells to be grown in the system are prokaryotic cells, such as bacterial cells. In some embodiments, the cells grown are eukaryotic cells, such as yeast cells, fungal cells, algae cells, plant cells, avian cells, or mammalian cells. The cells may be free floating in culture or may be adherent cells that adhere to one or more surfaces within the bioreactor. The cells may be transformed or otherwise engineered to produce a bioproduct (such as a heterologous protein, small molecule, or metabolite).

In some embodiments, the systems, devices, and methods described herein can be used in zero gravity or in microgravity conditions to allow cells to grow in zero gravity or microgravity conditions. Methods for constructing a bioreactor module

The systems, components and modules herein can be made of a variety of materials and these materials can be customized depending on the cells grown and the cell environment used. In some embodiments, components and modules or parts thereof are made by 3-D printing. Printing can use commercially available resins and ultraviolet (UV) curable biocompatible polymers. In some embodiments, the shapes of each mini module are discretely designed in a virtual environment. In some embodiments, components and modules are provided by commercially available components combined and configured together as described herein. In some embodiments, the biomaterial used may include a combination of three subcomponents (i.e., a biocompatible polymer, a photoinitiator, and a UV absorber).

The devices and systems of the present invention can be formed by 3-D printing (for example, stereolithography). In some examples, a computer-aided manufacturing (CAM) or computer-aided design (CAD) model of the device of the present invention is provided to a 3-D printing system using stereolithography, including providing a container with a resin, the resin including a photoinitiator and one or more polymer precursors. An ultraviolet (UV) laser can be used to draw a pre-programmed design or structure into the surface of the container with resin. The resin can be a photopolymer that is photochemically cured to form a single layer after contact with the UV laser. Additional resin can be added and cured in a layer-by-layer process. Stereolithography can be used to construct modules in a top-down or bottom-up additive manufacturing method.

Alternative methods for constructing reactor modules may include self-assembly of polymers (e.g., block copolymers) that form 3-D helical icosahedral structures, or subtractive manufacturing methods. Subtractive manufacturing methods may include chemical or mechanical removal of sacrificial materials. For example, sacrificial materials may be formed using adhesion manufacturing using a sintering laser. The sacrificial material may be immersed, impregnated, or otherwise coated in a biocompatible polymer. The sacrificial material may then be dissolved or mechanically removed to form the 3-D helical icosahedral structure from the biocompatible polymer. Customized Bioreactors and Systems

The bioreactor modules and systems herein can be designed and customized for specific cell types and specific bioproduct outputs. Specifically, production bioreactors can be designed for specific and personalized uses. The volume of the mini-modules, the channels within each mini-module, the number of mini-module levels, and the macrostructure can be selected to provide a cell environment (e.g., growth conditions) to achieve a given cell density, cell division rate, liquid shear forces, and the selection of an adaptive liquid medium, liquids for harvesting bioproducts (e.g., solvents for decomposing and/or distilling cell components), liquid flow rates, gas compositions, and gas flow rates.

In some embodiments, the system includes a mechanism for optimizing a bioreactor module and a cell environment incorporating one or more cells. The optimization mechanism may include a data loop that receives information about the cell environment (E) and cell behavior (B) from one or more sensors and cameras within the system. Additionally, for cells input into the system, genetic information (G) for the cell type may be input into the optimization mechanism to provide a relationship such as G*E=B.

In some embodiments, the cell environment is varied in one or more parameters (e.g., pH, liquid medium composition, gas composition, flow rate) and information about the cell environment is collected for each variation ( _E1 to _En ) and the resulting cell behavior ( _B1 to _Bn ) under each environmental variation. In some embodiments, the input cells vary (e.g., genetically distinct, e.g., cells varied in division rate, nutrient consumption, morphology, stress tolerance) and information about each cell is collected for each cell variation ( _G1 to _Gn ) and the resulting cell behavior ( _B1 to _Bn ) under the environment in the bioreactor. In some embodiments, variations in cell environment and cell genetics are used, and behavior is measured, such that the more variations in each cell (G ₁ to G _n ), the more the optimization mechanism measures behavior (B ₁ to B _n ) under more than one cell environment (E ₁ to _En ). The optimization mechanism ranks the G*E=B relationships across cell genetics and cell environments, indicates or otherwise compares the G*E=B relationships to provide information about the compatibility, optimization, and ranking of different cell genetics under various test cell environments. The optimization mechanism can thereby optimize production based on cell genetics, cell environment, or both. This allows the optimizer to provide parameters for optimizing a production bioreactor, including custom bioreactor design and custom cell environment conditions for the cell type and output to be produced.

In some embodiments, the optimization mechanism is a computer controlled device. In some embodiments, the optimization mechanism comprises a machine learning algorithm that uses data from one variation or set of variations to generate the next variation or set of variations to be tested. In some embodiments, the systems, devices, and methods described herein can be used in zero gravity or in microgravity conditions to grow cells in zero gravity or microgravity conditions. Example Bioreactor Modules and Systems

The bioreactor modules provided herein can be used separately or in combination to construct a system for producing, optimizing, and in some cases storing cells. In some embodiments, the systems, devices, and methods described herein can be used under zero gravity or under microgravity conditions so that cells grow in zero gravity or microgravity conditions. An exemplary embodiment of a 3-module system is shown in FIG1 . In this exemplary embodiment, three bioreactors are interconnected to the system: a cell chip 200, a sandbox bioreactor 400, and a production bioreactor 800. The liquid culture medium is mixed in a culture medium preparer 101 and moved for cleaning by an electroporator 20. The electroporator may include a cooler 21 to bring the temperature of the liquid culture medium to the operating temperature of the system. Pump 40 moves liquid medium from dispenser 101 into electroporator 20. Pump 50 flows medium through bubble trap 60 to remove bubbles generated by the electroporation process and bubble sensor 70 monitors the medium flowing therethrough for a correct bubble-free state. The medium then reaches container 30. In some embodiments, container 30 is comprised of two or more containers such that when full, a first container provides liquid medium to a bioreactor module while a second container is being filled. Each bioreactor module 200, 400, and 800 receives liquid medium from a container using pumps 100, 110, and 120, respectively. Gases are supplied to the bioreactor using a gas supply 500 which may include one or more gas storage devices for each gas component. The gas supply may include control devices for mixing and regulating the gas composition and gas flow to the bioreactor (including different mixtures and different flow rates to each bioreactor module).

Systems and bioreactors may also include specific output tanks and devices. Each bioreactor that includes liquid medium input and circulation may also include a fluid treatment device. A common fluid treatment device 600 may be used to collect spent liquid medium from all bioreactor modules. Similarly, for each bioreactor that includes gas input and circulation, a gas treatment device may collect spent and cell-secreted gas compositions. A common gas treatment device 550 may be used to collect output gases from all bioreactor modules.

The use of a bioreactor or in a system of multiple bioreactors includes one or more sensors to monitor one or more parameters including physical parameters, biological parameters and chemical parameters and combinations thereof. The example sensors shown in Figure 1 include a sensor 80 for monitoring a medium preparer, a sensor 81 for monitoring an electroporator, and a sensor 82 for monitoring one or more storage devices. An additional sensor 83 monitors the output from the cell chip module. Sensors 84 and 85 monitor the liquid and gas outputs, respectively, from the sandbox module. Similarly, sensors 86 and 87 monitor the liquid and gas outputs, respectively, from the production bioreactor module.

The system of one or more bioreactors may further include interconnections between bioreactor modules. As shown in the exemplary system of Figure 1, the cell chip is interconnected to the sandbox module by connector 280 and can be adjusted to allow cells to pass from the output of the cell chip to the input into the sandbox module. Connector 380 allows cells from the output of the sandbox module to flow to the input of the production bioreactor module. Connector 480 is an interconnection between the output of the production bioreactor to the collection tank or device collector 700. Collector 700 can collect cells, biological products, or a combination thereof from the production bioreactor 800. In some embodiments, collector 700 may include filters, membranes, or other units and/or modules for separation, such as separating cells from liquid medium, separating biological products from cells, and separating cell components or separating between cell components.

An exemplary cell environment 205 in a cell module 201 is shown in FIG2A. In this example, the cell chip includes a gate trap 220, a plurality of suction traps 240, and a plurality of overflow traps 260. A medium input port 202 for providing medium to the cell environment is at the input end. A collection port 210 for collecting cells growing in the cell chip module is at the opposite end (in the direction of liquid medium flow). Details of an exemplary medium loop 230 for the cell chip 201 are shown in FIG2B. The medium loop may include an input medium port 231, an output medium port 233, and a medium feeding channel 235. Details of an exemplary gas loop 250 for a cell chip 201 are shown in FIG2B. The gas loop may include an input gas loop 251, an output gas loop 253, and a gas distribution channel 255. FIG2C shows a side view of a cell chip 201 having an example of a configuration of a medium loop 230, a gas loop 250, and a cell environment 205.

FIG3A to FIG3F show an exemplary trap used with a cell chip module. An exemplary gate trap 221 shown near a medium input port 202 is diagrammatically shown in FIG3A (top view) and FIG3B (side view). The gate trap is composed of flow protectors 225 surrounding an interior space, with the protectors 225 having openings 227 therebetween that limit the flow of cells 203 out of the interior space of the trap. In some embodiments, the openings 227 between the protectors 225 are sized to be twice the diameter of the cells to be grown on the cell chip. In some embodiments, the opening 227 is sized to be larger than the diameter of the cells to be grown on the cell chip. The inoculation port 223 can be used to inoculate cells onto the chip and into the gate trap 221.

FIG. 3C and FIG. 3D show an exemplary overflow catcher 260 configuration from a top view (FIG. 3C) and a side view (FIG. 3D). The catchers are formed by a configuration of channelized box walls 245 configured to surround the interior space on three sides and create openings 247 between the wall segments during formation. The size of the openings 247 can be customized for the cells to be grown on the chip. In some embodiments, the diameter of the openings 247 is smaller than the diameter of live cells, but larger than the diameter of dead cells, so that live cells 203 remain trapped in the interior space of the channelized box walls 245 until they reach a number sufficient to overflow the box configuration, while dead cells pass through the openings 247 and are not retained in the interior space of the channelized box walls 245.

An exemplary suction trap 240 is shown in top and side views in Figures 3E and 3F. The traps have a porous field 262 and an ambient wall 264. The porous field is designed so that the size of the cells exceeds the pore diameter.

Figures 4A-4E show exemplary sandbox modules and components thereof. Figure 4A shows an exemplary sandbox segment 405. The exemplary segment includes a channel 445 in which cells and associated cell environments flow into a mixing module 420, where liquid medium from a channel 443 mixes with the cells and the mixture then flows into a cell chamber 410 for cell growth and, optionally, measurement by one or more sensors or cameras. Figure 4B shows an exemplary sandbox module cell environment 401, which includes multiple segments connected in series and parallel, cell inputs 440 and cell outputs 460 from the sandbox module. Each example segment includes a mixing module (e.g., 421, 422, 423) and a cell chamber (e.g., 411, 412, 413). FIG. 4C shows an example of a gas loop 470 (top) for an example sandbox cell environment 401 (bottom). FIG. 4D shows an example of a liquid medium loop 480 (top) with medium input and an example of a cell input channel 490 (bottom). FIG. 4E shows an example of a liquid medium loop 480, a gas loop 470, and a cell input channel 490 superimposed onto an example sandbox cell environment 401 to construct an example of an example sandbox module 400 shown from the top view and the side view.

5 shows an exemplary production bioreactor 800, which includes an inoculation port 802 for cells to enter the bioreactor, and a cell distribution structure 804 in which cells flow into the bioreactor and mix with liquid media and other cell environment components. Liquid media input 820 allows liquid media to be introduced into the production bioreactor, which is then distributed through media feed system 822 using media connections (e.g., 824), and unused media is collected 826. Gas enters at gas input 810 and is distributed throughout the bioreactor by gas phase channels 814, and is output through gas output 818. Gases and/or products produced by the cells can be collected through port 870.

Figures 6A-6C show examples of macrostructures used with the production bioreactors described herein. Figure 6A shows an example pyramid macrostructure 881 and a pyramid macrostructure stacked with a culture medium distribution system 883. Figure 6B shows example hollow pyramid macrostructures 885 and 887. Figure 6C shows an example log macrostructure 489.

In some embodiments, the macrostructure for producing a bioreactor is composed of mini-modules, each of which may have a given shape (such as a double helical icosahedron or a modified double helical icosahedron). FIG. 7A shows an exemplary embodiment of a modified double helical icosahedron shape 841 used to construct one or more mini-modules for producing a bioreactor. As shown in the bottom of the figure, the double helical icosahedron mini-module is schematically represented as a block 841A as shown in the macrostructure of FIGS. 6A to 6C. The modified double helical icosahedron shape 841 may include phases 842 and 844, which, when assembled (such as shown in FIG. 7B), may be assembled into stacked channels 843 and 845. Additionally, an inter-membrane space 846 is between phase 842 and phase 844. FIG. 7C shows an example sweep of a portion of an example channel 842 showing a constant diameter throughout the channel. Mini-Modules Assembled into Macrostructures

The mini-modules described herein can be assembled into macrostructures to provide calibrated control of medium and gas flow and distribution. In some embodiments, the mini-modules are assembled into macrostructures to produce a modified double helical tetrahedron (DG) that produces a bioreactor. Figures 9A to 9F illustrate an assembly starting with a first mini-module (e.g., DG) and assembling additional mini-modules so that the geometry is repeated to form a three-dimensional (3-D) matrix whose growth is limited to two of the three possible dimensions. The connection point from one mini-module to another mini-module is called a "mouth." This first assembly of interconnected mini-modules of the same orientation is called a "layer." The layers can be configured, for example, in a rhombus shape so that in some embodiments, if the same number of modules are connected in a selected direction, the resulting growth is not proportional and thus the growth of the layers is irregular relative to each other. In some embodiments, the layers are configured in a square shape or so that the resulting growth is proportional. Figures 9D-9F illustrate exemplary embodiments of layer assembly and growth.

At the edge of each layer, unconnected ports of the mini-modules can be used to connect the layer to other functionality such as input of medium flow or gas and output of spent medium, spent gas and output of cells and bioproducts produced by the cells.

In some embodiments, an assembly of layers of mini-modules ("first matrix") can be co-located with a second assembly of layers of mini-modules ("second matrix"), whereby the second matrix occupies the free space left by the first matrix, and whereby the matrices occupying the same volume have no touching points and maintain a constant minimum distance. An example assembly of two matrices is shown in Figures 10A to 10F. Figure 10A shows an example of a portion of a double helical icosahedron inscribed in a cube. Figure 10B shows orthogonal and cross-sectional views of the structure of Figure 10A. Figures 10C and 10D show examples of the growth direction of the second layer relative to the first layer. Figure 10E shows an example of a volume that is reduced from a pyramid and grows counterclockwise. Figure 10F shows an example of a macrostructure that grows clockwise along the vertical axis of a hollow pyramid.

In some embodiments, the mini-modules are assembled into a hollow pyramid macrostructure. The hollow pyramid has a hollow center and a volume of continuously increasing cross-section. Using the hollow pyramid macrostructure, the feed loop can serve both the outer periphery and the inner periphery. For the construction of the hollow pyramid, the matrix has an initial layer connected to the distributor and a layer connected in turn to the collector. The number of upper nozzles of the initial layer, like the number of lower nozzles of the layer connected to the collector, can belong to the set M = ²ⁿ . In this way, it is ensured that the connecting channels or trees can branch in pairs in a balanced manner. The tree can be a distributed structure (input) and collection (output) of a bubble-free bioreactor. In both the input and output, the channels can be transformed or branched from a single channel into ²ⁿ channels. The increase in volume between layers (which is the number of mini-modules added between one layer and the next in the flow direction) is determined by the bioreactor and is ordered by the following terms: (i) alternating growth meaning between the edges of its outer periphery; and (ii) increase in its inner periphery (i.e., the periphery of the inner hollow center). For example, if N is the number of modules in one of the edges of the outer periphery of the hollow pyramid, then n is the number of mini-modules constituting one of the edges of the inner periphery of the hollow pyramid, and then if N= (8;8) at one layer, then n=(4;4) (see Figure 12). This logic is repeated alternately between the outer edges of the pyramids in each layer and in a clockwise sense (with respect to the flow direction). The result is a stepped pyramid in which the steps form faceted spirals. The inner periphery also has spiral growth, but with a lower frequency than the outer periphery, and the growth direction of the inner periphery is opposite to that of the outer periphery (see Figures 13A and 13B). The interaction between the inner and outer spirals and the flow direction results in a vortex-like motion of the culture medium flowing within the hollow pyramid structure.

In some embodiments, the feed system is connected to the bioreactor through one or more subchannels. The subchannels of the feed loop may surround the perimeter of one or more layers in the bioreactor at equal distances on each side of a given layer. The subchannels may be connected to the edge of the layer at one or more mouths of the minimodule. Exemplary connector sets are shown in Figures 11A to 11F.

In some embodiments, the feed loop is connected to the bioreactor at 1, 2, 3, 4, 5, 6, 7, 8 or more points. In an embodiment, the feed loop serves a hollow pyramid macrostructure bioreactor and the feed loop has a division of 5 subchannels. One of these subchannels extends into the interior of the pyramid's internal channel and the remaining feed subchannels are parallel to the edges of the layers (external channels). The pressure and flow balance of the feed loop is maintained by the proportionality of the external and internal channels of the feed system. An exemplary feed loop for a hollow pyramid shape is shown in Figure 14.

In some embodiments, the macrostructure of the bioreactor is a sheet. In some embodiments, the feed system is connected to the bioreactor through one or more subchannels. The subchannels of the feed loop can surround the perimeter of one or more layers in the bioreactor at equal distances on each side of a given layer. The subchannels can be connected at one or more mouths of the minimodule as the edge of the layer. Exemplary connector sets are shown in Figures 11A to 11F.

In some embodiments, the bioreactor employs a thin sheet macrostructure composed of mini-modules (such as DG). The thin sheet macrostructure has a thin plate of constant thickness and an increasing cross-section including mini-modules. The constant thickness of the thin plate allows for uniform access to material from the feed loop. The increase in volume between layers (which is the number of modules added between one layer and the next in the flow direction) is determined by the bioreactor and ordered by alternating growth meaning between the shortest edges of the thin plates (e.g., see Figure 15). In a thin sheet macrostructure, there may be one or more thin plates, for example, 2, 3, 4, 5, 6, 7, 8 or more thin plates configured in parallel. The space between the sheets can be used to place the feed loop or parts of the modules in the feed sheets of the feed loop (see, for example, FIG. 16 ).

In some embodiments, the mini-modules are assembled into a tree (chessboard macrostructure) having at least one hollow row of constant cross-section that intersects the mini-module layers longitudinally. In some embodiments, the tree (chessboard macrostructure) has 1, 2, 3, 4 or more such rows. The rows can be used to provide areas for transporting liquid culture media and other substances through channels following the longitudinal rows. Collection of spent culture media, gases, cells and bioproducts can be performed on one or more or all of the external faces of the structure, driven by pressure differences between the center of the row and the face. An exemplary tree (chessboard macrostructure) is shown in Figure 17, and an exemplary feed and collection configuration is shown in Figure 18. Bioreactor connection system

Modules can be connected, coupled or fluidically connected by one or more connection systems. Figures 19A and 19B show an exemplary connection system, which includes a connector between a cell chip module and a fluid source or a fluid collection module. The connector includes a support and a set of hollow needles that allow fluids and/or cell-containing fluids to enter and flow out. In some embodiments, the connector is connected to a first module (such as a cell chip module) through the needles. The needles can be configured into several groups so that each group of needles can include a needle for inputting fluids and another needle for outputting fluids from the cell chip module. One end of the needle is used to enter a chamber or channel in the cell chip module and the other end of the needle can be connected to a fluid source, to a collection device or to another module.

In some embodiments, the set of needles includes at least one input needle and one output needle. In some embodiments, there are multiple sets of needles. Each set of needles can be guided to a separation chamber and/or a separation channel, and the fluid is guided to the separation chamber and/or the separation channel for input or removed for output.

In some embodiments, the connector can connect the cell chip module to one or more fluid sources (e.g., culture medium, nutrient supplements, chemical inputs, trypsin, wash/buffer solutions) that can be used to supply fluids to the cell chip module and remove spent fluids as needed. In some embodiments, the connector can connect the cell chip module to a second module (e.g., a sandbox bioreactor or a production bioreactor), such as for transferring cells from one module to another.

In an embodiment, the connection system includes a cleaning chamber so that the needle is cleaned and/or sterilized before entering a module (such as a cell chip module). In an embodiment, the cleaning chamber is one or more separate chambers at one end of the cell chip module. The cleaning chamber(s) are bounded on a first end by a partition that contains the cleaning chamber and protects it from the environment and through which the needle can penetrate from one end into the cleaning chamber. The cleaning chamber can be bounded on a second end by a safety membrane or other boundary that can contain a cleaning or sterilizing fluid (or gas) within the cleaning chamber. In these embodiments, the connector is connected to a fluid source at the other end of the needle, such as a fluid source having a cleaning or disinfectant and (several) cleaning solutions.

The channel is on the other side of the safety film or border. Once cleaned and sterilized, the needle can be placed in the channel through the safety film or border. The channel can be a medium channel that allows the medium to flow from the needle to other locations in the cell chip. The channel can be a cell collection channel, and cells present in the chip (such as cells grown and proliferated in the chip) can then be guided from the cell collection channel to the channel and then guided through the needle to the separation module or collection assembly. The channel can be a waste channel, and the used medium can be guided and removed from the chip through the waste channel.

Figure 19C shows an exemplary embodiment of the connections made by the connector system to the components containing the culture medium, disinfectant and to the waste collection and sandbox module. Connecting tubes or channels are connected from the connector system and valves are used to direct the fluid from the connector to the appropriate source, collector or module.

FIG. 19D shows an exemplary embodiment of a connection system having needles that penetrate a first chamber in a cell chip module, such as for cleaning and sterilization, illustrating an exemplary embodiment of the connection system during a cleaning procedure, having fluid flow from a component containing a sterilizing fluid to a sterilizing chamber in the cell chip and having one of each set of needles remove the used sterilizing fluid.

FIG. 19E shows an exemplary embodiment of a connection system with needles that penetrate a second chamber after cleaning/sterilization. The first set of needles (left) is positioned so that the input needle enters the medium channel/chamber and allows new medium to flow into the cell chip module. The middle set of needles is positioned so that one needle is positioned for exporting spent medium and culture waste from the channel in the cell chip module. The third set of needles (right) is positioned so that only the output needle enters the chamber/channel and is positioned for exporting medium and cells from the cell chip module. MODULE FOR ADHESIVE CELL CULTURE

In another aspect, the present invention provides a method for culturing cells. The method may include providing a plurality of cells to an adhesion bioreactor. The adhesion bioreactor may include at least one channel and a microporous membrane. The adhesion bioreactor may be part of a cell chip module or a sandbox bioreactor. At least a portion of the cells may be allowed to adhere to a surface of the at least one channel, such that the at least a portion of the plurality of cells grows and/or replicates on the surface of the channel to produce attached cells. A liquid culture medium may flow from the at least one channel through the microporous membrane. Flowing the liquid medium from the channel through the microporous membrane may wash the adherent cells (e.g., with fresh medium or sterile wash buffer), or may be used to detach the cells from the surface of the channel or may wash the detached cells. The detached cells (e.g., suspended cells) may be collected by flowing another liquid medium with the cells through the channel. The adherent bioreactor may be fluidly coupled to a cell chip module, a sandbox bioreactor, a bioreactor (e.g., a production bioreactor), or any combination thereof. The cell chip module may provide cells to the adherent bioreactor. Adhesion bioreactors can provide cells to a bioreactor (e.g., a production bioreactor).

Cells can be cultured or grown on a surface or artificial substrate under controlled conditions. For example, cells can be cultured in a monolayer or other surface growth configuration. FIG. 20 shows an exemplary layered module for adherent cell culture. FIG. 21A shows an embodiment of a module suitable for adherent cell culture. Cells can adhere to a material suitable for cell adhesion (such as PDMS). At a selected time point or a selected cell density, the adherent cells can be removed from the adhesion material and released into the surrounding culture medium, such as by adding an enzyme (such as trypsin) to a liquid flowing through the adherent (e.g., attached) cells so that the cells or parts thereof can be collected from the module.

In some embodiments, the adhesion material is delimited by one or more open channels, whereby the medium flows along with the adherent cells. The module may also contain boundaries (such as microporous membranes) that allow the medium or other fluids to pass through, but the cells cannot pass through the boundaries due to the size of the microporations.

In some embodiments, a module for adhering cells may have one or more chambers. An exemplary embodiment having an inoculation chamber, a filtration chamber, and an attachment chamber for importing cells into the module is shown in FIG. 20. The attachment chambers may have an adhesion material so that cells adhere to their surfaces.

Cells can be removed from an adhered site, for example, by including trypsin in a culture medium or other fluid that is passed through the adhered cells or incubated with the adhered cells. Once the cells are removed (e.g., from a PDMS material), the trypsin can be removed by washing with a culture medium or other fluid, and then passing such culture medium or fluid through a microporous membrane, thereby retaining the cells. The cells can then be resuspended in fresh culture medium or other fluid, and the flow of fluid through the channel can be used to move the cells or a portion thereof to a collection chamber or other output channel for collection.

Figures 21B to 21E show an exemplary series of cell attachment, trypsinization for cell detachment, and washing and collection. In some embodiments, the adherent cell module is a cell chip module or a portion thereof. In some embodiments, the adherent cell module is a sandbox module or a portion thereof. In some embodiments, the adherent cell module is part of a sandbox module or a portion thereof (such as one or more mini modules). Computer System

The present invention provides a computer system programmed to implement the methods of the present invention. FIG. 34 shows a computer system 3401 programmed or otherwise configured to control all internal processes of the programmed system, such as medium preparation and sterilization, flow control, gas flow rate, pressure and formulation, data acquisition through embedded sensors (e.g., physical, chemical and biological data), sensor data fusion and command control loops, image processing and generation of data sets associated with each process. Computer system 3401 can adjust various aspects of the microenvironmental conditions witnessed by cells within the system of the present invention, such as, for example, medium flow rate and subcomponent concentrations, mixing time, reservoir filling; pulse number, pulse voltage, duty cycle applied to the medium by the electroporator, gas flow and pressure, differential gas solution preparation, differential gas flow for some or all components, differential flow medium for some or all components; ... The computer system 3401 can be an electronic device of a user or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

Computer system 3401 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 3405, which may be a single-core or multi-core processor, or a plurality of processors for parallel processing. Computer system 3401 also includes memory or memory locations 3410 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 3415 (e.g., a hard drive), a communication interface 3420 for communicating with one or more other systems (e.g., a network adapter), and peripheral devices 3425 (such as cache, other memory, data storage, and/or electronic display adapters). Memory 3410, storage unit 3415, interface 3420, and peripheral device 3425 communicate with CPU 3405 via a communication bus (solid line) (such as a motherboard). Storage unit 3415 can be a data storage unit (or data storage library) for storing data. Computer system 3401 can be operatively coupled to a computer network ("network") 3430 with the help of communication interface 3420. Network 3430 can be the Internet and/or a business network, or an intranet and/or a business network that communicates with the Internet. In some cases, network 3430 is a telecommunications and/or data network. The network 3430 may include one or more computer servers that can implement distributed computing (such as cloud computing). In some cases, the network 3430, with the help of the computer system 3401, can implement a peer-to-peer network that enables devices coupled to the computer system 3401 to behave as clients or servers.

The CPU 3405 may execute a sequence of machine-readable instructions that may be embodied in a program or software. The instructions may be stored in a memory location such as the memory 3410. Instructions may be directed to the CPU 3405, which may then program or otherwise configure the CPU 3405 to implement the methods of the present invention. Examples of operations performed by the CPU 3405 may include fetch, decode, execute, and write back.

CPU 3405 may be part of a circuit, such as an integrated circuit. One or more other components of system 3401 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

Storage unit 3415 can store files (such as drivers, libraries, and saved programs). Storage unit 3415 can store user data (such as user preferences and user programs). In some cases, computer system 3401 can include one or more additional data storage units external to computer system 3401 (such as located on a remote server that communicates with computer system 3401 through an intranet or the Internet).

Computer system 3401 can communicate with one or more remote computer systems via network 3430. For example, computer system 3401 can communicate with a user's remote computer system (e.g., a virtual private network, a computer hosted in a service such as Amazon Web Services (AWS), satellite communications). Examples of remote computer systems include: a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone, Android enabled device, Blackberry®), or a personal digital assistant. A user can access computer system 3401 via network 3430.

Methods as described herein may be implemented by machine (e.g., computer processor) executable program code stored on an electronic storage location of the computer system 3401, such as on the memory 3410 or electronic storage unit 3415, for example. The machine executable or machine readable program code may be provided in the form of software. During use, the program code may be executed by the processor 3405. In some cases, the program code may be retrieved from the storage unit 3415 and stored on the memory 3410 for ready access by the processor 3405. In some scenarios, the electronic storage unit 3415 may be eliminated, and the machine executable instructions are stored on the memory 3410.

The program code may be precompiled and configured for use with a machine having a processor adapted to execute the program code, or may be compiled during runtime. The program code may be supplied in a programming language which may be selected to enable the code to be executed in a precompiled or compiled manner.

Aspects of the systems and methods (such as computer system 3401) provided herein may be embodied in a programmatic manner. Various aspects of the technology may be considered as a "product" or "article of manufacture" in the form of machine (or processor) executable program code and/or associated data (which is carried in or embodied in a machine-readable medium type). The machine-executable program code may be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard drive. "Storage" type media may include any or all of the tangible memory of a computer, processor or the like, or its associated modules (such as various semiconductor memories, tape drives, disk drives and the like) which may readily provide non-transitory storage for software programming. All or part of the software may sometimes be communicated over the Internet or various other telecommunications networks. Such communications may enable, for example, the software to be loaded from one computer or processor to another, such as from a management server or mainframe computer to an application server's computer platform. Thus, another type of media that can carry software components includes optical, radio, and electromagnetic waves, such as those used over wired and optical fixed-line networks and across physical interfaces between local devices via various airlinks. Physical components that carry these waves (such as wired or wireless links, optical links, or the like) can also be considered media that carry software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any media that participates in providing instructions to a processor for execution.

Thus, machine-readable media (such as computer executable code) can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. For example, non-volatile storage media include optical and magnetic disks, such as those used to implement the databases shown in the diagrams, or any of the storage devices in any computer or the like. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire, and optical fibers (including the wires that comprise the bus within a computer system). The carrier transmission medium may take the form of an electrical or electromagnetic signal or an acoustic or light wave, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card tapes, any other physical storage media with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transmits data or instructions, a cable or link that transmits such a carrier wave, or any other medium from which a computer can read the program code and/or data. Many such forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system 3401 may include or communicate with an electronic display 3435 including a user interface (UI) 3440 for providing capabilities such as setup, bioprocess reports listing measured variables at each stage of the system in real time, exporting and importing files (e.g., configuration files, updates), calibration, alarms (e.g., errors, maintenance, replacement of consumables). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

The methods and systems of the present invention may be implemented by one or more algorithms. The algorithms may be implemented by software after being executed by the central processing unit 3405. For example, the algorithms may use feedback loops to adjust variables of the control system; detect problems in the process by image recognition and pattern analysis, fuzzy logic, and hard and soft limit enforcement; correlate specific and non-specific data through machine learning (e.g., supervised, unsupervised, and/or reinforced) to optimize process conditions, process results, modeled behavior, and simulations within the system. Examples Example 1 : Simulating Fluid Flow and Mixing in Layers of Mini-Modules

Considering that the flow is laminar, the simulation can be solved in two cases; on the one hand the velocity field and on the other hand the advective diffusion transport of the microorganisms to be analyzed. In this case, it was decided to use two species (S1 and S2) that enter the inlet of the module or each branch of the inlet of the different coupled modules at a specific concentration. For S1 the diffusion value is established according to the diffusion coefficient of fluorescein in water and for S2 according to its diffusion coefficient reduced by 2 orders of magnitude.

The development and mixing process between the flows was simulated using a constant average inlet rate of 5 μm/sec and the concentration of the species was followed from the outlet over the entire cross section. Assuming that the flow rates for both inlet branches were equal, a concentration of 50% of the inlet value expected from good mixing was obtained.

In FIG. 22A , the cross-sectional view of a single mini-module shows poor or low mixing caused by the low diffusion coefficient of the species, while in FIG. 22B , the diffusion coefficient is higher and successful mixing can be understood. FIG. 23A-B show the results of a combination of ten mini-modules subjected to the simulation procedure. The pure distribution (representing cells) and the concentration of the medium (color) are shown. On the one hand, this particular distribution of mini-modules can be observed, where cells enter from the top and the medium enters through the sides, the cells do not leave at all lower outlets and the medium is not evenly distributed (due to the short flow path in the simulation). Figures 25A-25C show another configuration of mini-module simulations performed with 6 module levels. In this configuration, the first level starts with a larger number of modules, so that the percentage of modules in each level increases less (1 module row per row/layer) and different media are fed into the array with fewer nozzles. In this configuration, the distribution of cells and the distribution of media culture are both reasonably uniform. Example 2 : Constructing a macroscopic structure of mini-modules

The bioreactor was designed using the macrostructure shown in Figure 24 consisting of layers of DG mini-modules and with the feed loop as shown. All systems and connections were 3-D printed using an SLA 3-D printer (Peopoly Moai) using commercial resin. The printed bioreactor is shown as a cross-section in Figures 23A and 23B.

Additional bioreactors were printed using an SLA printer and photocurable resins. The design consists of two phases in "positive form" (the volumes of the two phases are entangled with each other without an intermediate membrane). Figure 25A shows an isometric view of the test file used, which consists of several layers of double helical tetrahedra (diameter: 600 micrometers (μm)) and a solid substrate for better manipulation. It should be noted that no feed system for the phases was placed. Figure 25B shows a successfully printed test file on PEGDA photocurable resin and Figure 25C shows a successfully printed test file using a commercial resin. Example 3 : Demonstration of fluid flow through a mini module

A direct light projection type 3-D printer using commercial resin was used to print a matrix consisting of 4 layers and rows of 4x2 modules with input/output connections, where the diameter of the helical icosahedron was 500 μm. The printed bioreactor was injected with red dye at one input connection. Figure 26A shows the loop filled with red dye. A second print consisting of the first matrix and the second matrix was performed to form a double helical icosahedron, both helical icosahedrons having a diameter of 500 μm. One matrix was injected with red dye at the input connection and the other matrix was injected with blue dye. Figure 26B shows the 2 matrices differentiated by color. Example 4 : Strains on a chip

An example strain on a chip embodiment constructed using the design is shown in Figure 27. Figure 28 shows the progression of images obtained in the assay over time. To simulate microorganisms, glass particles with an approximate average diameter of 50 microns were introduced into the structure as follows.

With port 2 closed, a flow of distilled water is applied from port 3 to port 1. In the initial stage, the loop is filled with freeze-dried water for simulation; for microbial inoculation, the loop can first be filled with culture medium. The collection output (2) is closed and the chip is "inoculated" with glass particles to simulate microorganisms. The flow action transports the particles to the chamber, and once port 2 is opened, the suction effect through the porous membrane can keep most particles in place.

The loop has input ports 3 to 4 and an output for the medium (1). With this configuration, the primary loop is seeded through port 3, combined with the introduction of liquid medium (here water) into port 4. Pressure balance forces the medium to occupy the secondary loop and leave the loop through output port 1. The porous membrane acts as a filter, trapping the simulated microorganisms in the primary loop chamber.

Once the chamber is full, the inoculation port can be closed and the collection output enabled. Due to the dynamics of the flow through the chamber, the velocity of the particles and the impacts on the particles in the chamber decrease dramatically as they move away from the center axis (see Figure 28). This increases the pumping effect of the secondary loop, causing the microbial population residing in the chamber to continue to proliferate (when using living organisms instead of the simulation used in this example). As the number of microorganisms increases, some of the microorganisms shift to a specific point near the center axis of the flow from port 4 and are dragged to port 2 (see Figure 28). Figure 29 shows the movement of ions over time. Example 5 : Example Sandbox Bioreactor Unit

Figure 30A shows the design of an exemplary sandbox unit with a mixing module, a single growth chamber, and a collection port. Figures 30B to 30D show constructed units made of PDMS. In Figure 30B, water (colorless) is inoculated into the center channel at a flow rate of 985 microliters (μL) per hour, and water with blue dye is inoculated into each side channel at a flow rate of 985 μL per hour. In Figure 30C, water (colorless) is inoculated into the center channel at a flow rate of 984 μL per hour, and water with blue dye is inoculated into each side channel at a flow rate of 3335 μL per hour. Figure 30C shows a module inoculated with an unbalanced flow between side channels. Example 6 : Constructing a sandbox bioreactor with multiple interconnected units

Figure 31 shows the design of the culture and gas loops for an exemplary sandbox bioreactor module. The three layers of the module share a series of through holes in which screws, washers and nuts are adjusted to prevent leaks.

The culture medium distribution layer in this module is a composite distribution system formed with hoses. The hoses can have a diameter between 50 microns and 500 microns and can be formed from biocompatible materials. The length of the hoses is adjusted by calculating the load loss of the culture medium and comparing the load loss with the pressure in each mixing module. The PDMS layer separates the gas layer and the culture medium layer. Figure 32 shows the printed culture medium layer of the sandbox. Figure 33 shows the assembled layers. Water with blue dye is used to inoculate the sandbox from the input port to show flow through the sandbox module.

Although preferred embodiments of the present invention have been shown and described herein, it will be understood by those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be interpreted in a restrictive sense. Those skilled in the art will now think of several variations, changes and substitutions that do not deviate from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions that are dependent on various conditions and variables described herein. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. Therefore, it is contemplated that the present invention should also cover any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the invention and to cover thereby methods and structures within the scope of these claims and their equivalents.

20: Electroporator 21: Cooler 30: Container 40: Pump 50: Pump 60: Bubble Trap 70: Bubble Sensor 80: Sensor 81: Sensor 82: Sensor 83: Sensor 84: Sensor 85: Sensor 86: Sensor 87: Sensor 100: Pump 101: Medium Preparer/Preparer 11 0: Pump 120: Pump 200: Cell chip/bioreactor module 201: Cell module/cell chip 202: Culture medium input port 203: Cell/living cell 205: Cell environment 210: Collection port 220: Gate trap 221: Gate trap 223: Inoculation port 225: Flow protector/protector 227: Opening 230 :Cell chip culture medium loop/culture medium loop 231: Culture medium input port 233: Culture medium output port 235: Culture medium feed channel 240: Suction trap 245: Channelized box wall 247: Opening 250: Gas loop 251: Gas input loop 253: Gas output loop 255: Gas distribution channel 260: Overflow trap 2 62: porous field 264: environmental wall 280: connector 380: connector 400: sandbox bioreactor/bioreactor module/sandbox module 401: sandbox module cell environment/sandbox cell environment 405: sandbox fragment 410: cell chamber 411: cell chamber 412: cell chamber 413: cell chamber 420: hybrid module 421: Mixing module 422: Mixing module 423: Mixing module 440: Cell input 443: Channel 445: Channel 460: Cell output 470: Gas loop 480: Connector/liquid medium loop 489: Example log macrostructure 490: Cell input channel 500: Gas supply 550: Gas processor 600: Fluid handler 700: Collection tank or device collector/collector 800: Production bioreactor/bioreactor module 802: Inoculation port 804: Cell distribution structure 810: Gas input port 814: Gas phase channel 818: Gas output port 820: Liquid culture medium input port 822: Culture medium feeding system 824: Culture medium connection Connector 826: Collection 841: Modified double helical tetrahedron shape 841A: Block 842: Phase/channel 843: Channel 844: Phase 845: Channel 846: Inter-diaphragm space 870: Port 881: Pyramidal macrostructure 883: Culture medium distribution system 885: Hollow pyramidal macrostructure 887: Hollow pyramidal macrostructure Structure 3401: Computer system / System 3405: Central processing unit (CPU) / Processor 3410: Memory or memory location 3415: Electronic storage unit / Storage unit 3420: Communication interface / Interface 3425: Peripheral device 3430: Computer network / Network 3435: Electronic display 3440: User interface (UI)

The novel features of the present invention are specifically described in the accompanying claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings (also referred to herein as "figures" and "FIG.") which illustrate illustrative embodiments in which the principles of the present invention are utilized; in the accompanying drawings:

FIG. 1 shows an exemplary embodiment of a system having three modules;

FIG. 2A shows an exemplary embodiment of a cell chip module 201; FIG. 2B shows an exemplary embodiment of a cell environment 205, a cell chip culture medium circuit 230, and a cell chip gas circuit 250 as individual "layers" of a cell chip; FIG. 2C shows the side profile of the layers of the cell chip;

3A to 3F provide an exemplary embodiment of a cell trap for use with a cell chip module; FIGS. 3A to 3B show an exemplary embodiment of a gate trap; FIGS. 3C to 3D show an exemplary embodiment of an overflow trap; and FIGS. 3E to 3F show an exemplary embodiment of a suction trap;

4A to 4E provide exemplary embodiments of a sandbox bioreactor;

FIG. 5 provides an exemplary embodiment of a production bioreactor;

6A to 6C provide exemplary embodiments of macrostructures for producing bioreactors;

FIG. 7A shows an example embodiment of a modified double helical icosahedron shape or structure and assembly into overlapping channels such as for use in producing a bioreactor as described herein; FIG. 7B provides an example of a modified double helical icosahedron shape or structure assembled into two overlapping channel networks; FIG. 7C provides an example of curvature produced by assembling a modified double helical icosahedron into a channel;

FIG8 shows a scheme of exemplary methods for cell growth, storage, environmental optimization, and scale-up production;

9A to 9F show an exemplary scheme for assembling mini-modules into a macrostructure; FIG. 9A shows an example of a mini-module; FIG. 9B shows an example of assembling mini-modules into an exemplary three-dimensional matrix; FIG. 9C shows an exemplary three-dimensional matrix; FIG. 9D shows an exemplary layer of a three-dimensional matrix; and FIG. 9E to 9F show an exemplary assembly including a plurality of three-dimensional layers;

10A to 10F show examples of layer assemblies of various shapes (e.g., square and square-like assembly shapes);

11A to 11F show examples of module layers connected to an example feedback loop;

FIG. 12 shows an example layer of a hollow pyramid shape;

13A and 13B provide examples for the growth of hollow pyramid shapes;

FIG. 14 shows an example of an external feed loop for a hollow pyramid shape;

FIG15 shows an example of a thin slice macrostructure;

FIG16 shows an example of a thin-film macrostructure with a feed loop;

FIG17 shows an example macrostructure;

FIG18 shows an example feed and collect configuration;

19A to 19E show example connection systems; FIG. 19A shows an overview of an example connection system including a connector between a cell chip module and a fluid source; FIG. 19B shows an example connection system with input and output needles; FIG. 19C shows an example connection made by an example connector system; FIG. 19D shows an example embodiment of a connection system with needles penetrating a chamber in an example cell chip module; FIG. 19E shows an example connection system with needles penetrating a second chamber;

FIG. 20 shows an example multi-layer module for adherent cells;

FIG. 21A shows an example of a layer module for adherent cells; FIG. 21B shows an example of cells attached to a layered structure; FIG. 21C shows an example of trypsin washing for cell detachment; FIG. 21D shows an example of cell detachment; FIG. 21E shows an example of flow through detached cells;

FIG. 22A shows a cross-sectional view of an example mini-module; FIG. 22B shows a cross-sectional view of an example mini-module with increased mixing;

FIG. 23A shows an example combination of ten mini-modules; FIG. 23B shows an example combination of ten mini-modules with increased mixing;

FIG. 24 shows an exemplary bioreactor design with a macrostructure;

FIG. 25A shows an isometric view of an example macrostructure; FIG. 25B shows an example printed macrostructure; FIG. 25C shows an example printed macrostructure made from a commercial resin;

FIG. 26A shows an exemplary bioreactor loop filled with dye; FIG. 26B shows an exemplary double helical tetrahedron structure filled with dye;

FIG. 27 shows an example design for an example strain on a chip;

FIG28 shows an example of particle flow in a chamber;

Figure 29 shows an example of particle flow in time progression;

FIG. 30A shows an exemplary sandbox unit having a hybrid module; FIGS. 30B to 30D show an exemplary sandbox unit formed of polydimethylsiloxane;

FIG. 31 shows the gas loop and culture medium loop for an exemplary sandbox bioreactor;

FIG. 32 shows an exemplary printed culture layer of a sandbox;

FIG. 33 shows the assembled layers of an example sandbox; and

FIG. 34 shows a computer system programmed or otherwise configured to implement the methods provided herein.

800: Production of bioreactors/bioreactor modules

802: Immunization port

804:Cell allocation structure

810: Gas input port

814: Gas phase channel

818: Gas output terminal

820: Liquid culture medium input port

822: Culture medium feeding system

824: Culture medium connector

826: Collection

870: Port

Claims (15)

A bioreactor, comprising: an inlet configured to receive a plurality of cells; a plurality of mini-modules, which are fluidically connected to the inlet, wherein the mini-modules of the plurality of mini-modules include a double helical tetrahedron structure or a modified double helical tetrahedron structure, wherein the plurality of mini-modules are fluidically interconnected to provide at least one microchannel configured to allow the plurality of cells to flow; and an outlet, which is fluidically connected to the plurality of mini-modules, wherein the outlet is configured to guide the plurality of cells or their derivatives out of the at least one microchannel. A bioreactor as claimed in claim 1, wherein the plurality of mini-modules are interconnected in a manner to provide at least two non-overlapping microchannels each having a constant average curvature. A bioreactor as claimed in claim 2, wherein the first microchannel of the at least two non-overlapping microchannels is configured to allow a liquid culture medium to flow, and wherein the second microchannel of the at least two non-overlapping microchannels is configured to allow a gas combination to flow. A bioreactor as claimed in claim 3, wherein the first microchannel and the second microchannel of the at least two non-overlapping microchannels are separated by a porous membrane. A bioreactor as claimed in claim 4, wherein the area of the first microchannel is equal to the area of the second microchannel, and wherein the area of the porous membrane is the sum of the areas of the first microchannel and the second microchannel. A bioreactor as claimed in any one of claims 1 to 5, wherein the plurality of mini-modules are assembled into a macroscopic structure, the macroscopic structure being selected from the group consisting of pyramids, hollow pyramids, thin pyramids, thin sheets, chessboard arrangements and logs. A bioreactor as claimed in claim 6, wherein the plurality of mini-modules are arranged in layers within the macrostructure, and wherein the layers are configured so that the velocity of the liquid culture medium in each layer is substantially the same. A bioreactor as claimed in any one of claims 1 to 5, wherein the liquid culture medium flowing through the at least one microchannel has a velocity greater than the free fall velocity of the cells flowing through the at least one microchannel. A bioreactor as claimed in claim 7, further comprising a medium input device configured to flow liquid medium to each of the layers in the macrostructure, and wherein the volume of liquid medium provided to each layer by the liquid medium device maintains a substantially constant cell density in each of the layers. A bioreactor as claimed in claim 7, wherein the rate of liquid culture medium passing through each mini-module is determined by the cell division rate, so that the time it takes for a cell to pass through a single mini-module or a layer of mini-modules is substantially the same as the cell division rate. A method for mass production of cells, comprising: introducing a first plurality of cells into the inlet of a bioreactor as claimed in any one of claims 1 to 10; supplying a gas composition into the bioreactor; and growing the cells under conditions of cell division to produce a second plurality of cells; wherein the second plurality of cells are transferred between a module of the plurality of mini-modules and another module of the plurality of mini-modules, and wherein the second plurality of cells are transferred from the inlet end of the bioreactor to the outlet end of the bioreactor. The method of claim 11, further comprising collecting the second plurality of cells or a portion thereof from the outlet. The method of claim 11 or 12, wherein the first plurality of cells include chimeric antigen receptor T (CAR-T) cells, stem cells, recombinant cells or differentiated cells. The method of claim 11 or 12, wherein the first plurality of cells comprises bacterial cells, fungal cells, yeast cells, eukaryotic cells, plant cells or algae cells. The method of claim 11 or 12, wherein the second plurality of cells produces at least one bioproduct, and wherein the at least one bioproduct is collected from the bioreactor.